Apparatus and method for imaging small objects in a flow stream using optical tomography

• US 6,522,775 B2
• Filed: 08/10/2001
• Issued: 02/18/2003
• Est. Priority Date: 03/28/2001
• Status: Expired due to Term

• Current Assignee: VisionGate, Inc.
• Original Assignee: Alan C. Nelson
• Inventors: Nelson, Alan C.
• Primary Examiner(s): Johns, Andrew W.
• Assistant Examiner(s): NAKHJAVAN, SHERVIN K
• Application Number:
  US09/927,151
• Publication Number:
  US 20020141625A1
• Time in Patent Office: 557 Days
• Field of Search: 382/131, 382/133, 250/455.11, 250/461.2, 353/28, 378/8, 378/11, 378/23, 378/25, 377/10
• US Class Current: 382/133
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 2223/419 computed tomograph
• CPC Class Codes: G01N 23/046 using tomography, e.g. computed tomography [CT]
• CPC Class Codes: G06T 11/006 Inverse problem, transformation from projection-space into object-space, e.g. transform methods, back-projection, algebraic methods

Abstract

A flow optical tomography system includes a flow cytometer, and at least one reconstruction cylinder positioned around a capillary tube. A photon source and a photon sensor work together with a pulse height analyzer to provide a first trigger point for the beginning of an object or cell, and a second trigger point for the end of the cell. The trigger signal is received by the reconstruction cylinder. The reconstruction cylinder includes optical point sources having a selectable emission wavelength, disposed in a geometric pattern around the cylinder perpendicular to and concentric with the capillary tube axis that facilitate the acquisition of transmitted, attenuated projection images of the flowing cells. The sensors also collect projections of fluorescence emitted from tagged molecular probes associated with nuclear and/or cytoplasmic structures or cell membranes. The projections are algorithmically processed to provide three dimensional information about the cells and their disease state.

Claims

1. A flow optical tomography method for imaging and analysis of microscopic objects, the method comprising the steps of:

(a) injecting at least one object into an injection tube;

(b) controlling the flow of the at least one object through a capillary tube such that the at least one object elongates along an axis of flow and moves proximately along a central axis of the capillary tube;

(c) sampling with at least one optical point source, located around a circumference of the capillary tube, in conjunction with at least one opposing optical sensor disposed opposite the at least one optical point source at a distance from the capillary tube such that there is no focal plane, and where multiple projection angles through the at least one object are sampled as it flows past the at least one optical point source and at least one opposing optical sensor; and

(d) generating a series of timing signals, such that each timing signal coincides with a specific position along the axis in the z-direction of the at least one object, so as to generate a set of timed optical projections through the at least one object.

2. The method of claim 1 further comprising the step of reconstructing the set of timed optical projections to form a two dimensional (2D) slice.

3. The method of claim 2 further comprising the step of combining sequential 2D slices to produce a three dimensional (3D) image of the at least one object.

4. The method of claim 3, wherein the at least one object comprises a cell including at least one molecular probe, where the molecular probe provides molecular probe information, the method further comprising the step of combining the 3D image and the molecular probe information so as to ascertain any association between particular sub-cellar structures and the at least one molecular probe.

5. The method of claim 2, wherein the at least one object includes a cell having at least one molecular probe therein, where the molecular probe provides molecular probe information, the method further comprising the step of combining the 2D slice and the molecular probe information so as to ascertain any association between particular subcellar structures and the at least one molecular probe.

6. The method of claim 1 further comprising the step of reconstructing the set of timed optical projections to form a three dimensional (3D) volume.

7. The method of claim 6, wherein the at least one object comprises a cell including at least one molecular probe, where the molecular probe provides molecular probe information, the method further comprising the step of combining the 3D volume and the molecular probe information so as to ascertain any association between particular sub-cellar structures and the at least one molecular probe.

8. The method of claim 1, wherein the set of timed optical projections comprise two dimensional (2D) optical projections, further comprising the step of producing a three dimensional (3D) image of the at least one object from two dimensional (2D) optical projections.

9. The method of claim 8 further comprising the step of using the 3D image to yield quantitative measures of microscopic structures.

10. The method of claim 9 wherein the microscopic structures comprise sub-cellular structures.

11. The method of claim 8 wherein the at least one object includes a cell having at least one tagged molecular probe therein, further comprising the step of using the 3D image to determine a location and amount of the at least one tagged molecular probe.

12. The method of claim 8, wherein the at least one object comprises a cell including at least one molecular probe, where the molecular probe provides molecular probe information, the method further comprising the step of combining the 3D image and the molecular probe information so as to ascertain any association between particular sub-cellar structures and the at least one molecular probe.

13. The method of claim 1 further comprising the step of creating laminar flow within the capillary tube such that the at least one object moves with a constant velocity.

14. The method of claim 1 wherein the at least one object is a cell.

15. The method of claim 14 further comprising the step of processing and analyzing the projection images directly to assess the disease status or transformation state of a cell.

16. The method of claim 1, wherein the at least one object includes a cell, the method further comprising the step of operating in a circle of reconstruction for cell imaging and analysis, such that the space being sampled by projections is modeled as consisting of at least three compartments:

(a) the fluid outside the cell,

(b) the cell cytoplasm, and

(c) the cell nucleus.

17. The method of claim 16, wherein at least one molecular probe is bound to the cell, further comprising the steps of:

(a) computing boundary surfaces of the cell including a cell wall and a nuclear wall, if certain ones of the at least one molecular probe bind only to surfaces of the cell wall and the nuclear wall; and

(b) otherwise characterizing the surfaces of the cell wall and the nuclear wall as transition surfaces between the at least three compartments.

18. The method of claim 16 further comprising the steps of:

(a) measuring the relative over or under expression of a gene product in the cell cytoplasm relative to the nucleus; and

(b) normalizing for non-bound probes in the background suspension fluid.

19. The method of claim 18 further comprising the step of using a tagged antibody probe to assess at least one of a disease state and a transformation state of the cell if the gene product is a protein.

20. The method of claim 18 further comprising the step of using a nucleic acid probe to assess at least one of a disease state and a transformation state of the cell if the gene product is a protein.

21. The method of claim 1 further comprising the step of illuminating the at least one object with an intense white light source and simultaneously collecting multiple filtered bandwidths.

22. The method of claim 1 further comprising the steps of

(a) tagging molecular probes with a reporter that emits light of a different wavelength when stimulated by a primary source of photons;

and

(b) filtering a secondary emission from the reporter to separate the primary source photons from the secondary emission photons.

23. A flow optical tomography system for imaging and analysis of microscopic objects, comprising:

(a) a flow cytometer including a capillary tube having a central flow axis;

(b) at least one reconstruction cylinder positioned around the capillary tube; and

(c) a triggering device, located to view at least one object flowing through the capillary tube, for creating a trigger signal for the at least one object, where the trigger signal is received by the at least one reconstruction cylinder, and where the at least one reconstruction cylinder responds to the trigger signal by producing signals representing a projection image about the at least one object and the projection image signals are processed to provide three dimensional information about the at least one object.

24. The flow optical tomography system of claim 23, where the at least one object moves with a velocity (V) through the capillary tube, and wherein the at least one object comprises a cell having wall of cell cytoplasm and a wall of cell nucleus, further, during the course of flowing through the capillary tube, the cell passes through a plurality of reconstruction planes, a planar slice through the wall of the cell nucleus lies within each reconstruction plane, where, a distance (d) between reconstruction planes is typically less than 10 microns and a point within the cell coincides with each reconstruction plane at time intervals (t), where the time intervals are described according to the relationship:

25. The flow optical tomography system of claim 24, where the cell is labeled with at least one tagged molecular probe for disease diagnosis.

26. The flow optical tomography system of claim 23, further comprising:

(a) means for controlling the velocity of the at least one object flowing proximately along an axis;

(b) means for locating two dimensional (2D) planes of reconstruction along an axis of the at least one object to create a three dimensional (3D) image of the at least one object; and

(c) means for correctly locating the position of the at least one object in the reconstruction cylinder to create a three dimensional (3D) image of the cell from a set or plurality of sets of two dimensional (2D) projection data.

27. The system of claim 26 further comprising a means for processing and analyzing a set of projection images directly to assess the disease status or transformation state of a cell.

28. The system of claim 23 wherein the at least one reconstruction cylinder comprises:

(a) a helix of point sources disposed at a predetermined helical pitch, where each point source generates a beam of photons, where the beam has a fan or cone shape, where the at least one object moves past the helix of point sources to generate multiple projections at different angular orientations through the at least one object;

(b) a locus of point sources disposed in a geometric pattern around the at least one reconstruction cylinder concentric with a capillary tube axis; and

(c) where each of the helix of point sources emits photons that pass as radiating projections through the at least one object and are detected by at least one sensor opposite each of the helix of point sources.

29. The system of claim 28 wherein the at least one reconstruction cylinder includes a plurality of reconstruction cylinders arranged in series, and where the helix of point sources illuminating the at least one object passing through the flow tube emit at wavelengths spanning the electromagnetic spectrum from x-ray to far infrared.

30. The system of claim 29 wherein the helix of point sources includes sources within and/or between the plurality of reconstruction cylinders that differ in their emission spectra.

31. The system of claim 30 wherein the emission spectra comprise narrow band spectra centered around the excitation maxima of immunofluorescence dyes and tags.

32. The system of claim 29 wherein the at least one sensor comprises light sensor arrays spectrally bandpass filtered for sensitivity to the wavelengths emitted by fluorophores employed for at least one of immunofluorescence studies and ploidy studies.

33. The system of claim 29 wherein the plurality of reconstruction cylinders are arranged in series with intervening sections of capillary tube.

34. The system of claim 29 wherein the range of the emitted wavelengths is limited to span the electromagnetic spectrum from 10 Angstroms to 2000 microns.

35. The system of claim 23 wherein the at least one reconstruction cylinder includes a plurality of point sources arranged along a geometric pattern on the capillary tube such that each point in the at least one object is sampled from a multitude of angles as it passes through the plurality of point sources.

36. The system of claim 35 wherein the geometric pattern comprises a helical pattern.

37. The system of claim 36 where the plurality of point sources cover any angular extent of the circumference and are spaced in equiangular increments along at least 180 degrees.

38. The system of claim 36 wherein the at least one reconstruction cylinder further comprises a plurality of fixed point sources having successive subsets of elements staggered to align properly with each different point source along the helical pattern.

39. The system of claim 36 wherein the at least one reconstruction cylinder includes an array of sensing elements curved along a cylindrical circumference that is concentric with the at least one reconstruction cylinder.

40. The system of claim 23 wherein the capillary tube comprises uniformly thin walls relative to the cross-sectional area of the capillary flow.

41. The system of claim 23 wherein the triggering device is located upstream from the at least one reconstruction cylinder to provide a timing signal to initiate and subsequently terminate data collection as the at least one object enters then emerges from the at least one reconstruction cylinder.

42. The system of claim 23 wherein the triggering device comprises elements selected from the group consisting of a laser diode, CCD, PMT, a solid state photodetector and combinations of the forgoing elements.

43. The system of claim 23 wherein the triggering device generates a trigger signal that, in conjunction with the at least one object velocity, is used to calculate when the downstream at least one reconstruction cylinder can commence data collection for the at least one object of interest.

44. The system of claim 43 wherein the triggering device generates a trigger signal that, in conjunction with the at least one object velocity, is used to calculate a point in time for the downstream at least one reconstruction cylinder to proceed to acquire multiple sets of projection data at temporal increments based on the set of upstream trigger signals.

45. The system of claim 23 wherein the capillary tube is constructed to produce velocities in the range of 1 meter/sec to 10 meters/sec.

46. The system of claim 23 wherein the at least one reconstruction cylinder produces projection rays from a plurality of fixed point sources into the capillary tube, and photons emitted from the plurality of fixed point sources have a selected projection geometry such that there is no focal plane.

47. The system of claim 46 wherein the selected projection geometry is selected from the group consisting of a cone shape and a fan shape.

48. The system of claim 46 wherein the projection rays pass through at least one object to be detected by at least one array of sensing elements.

49. The system of claim 48 wherein:

(a) the at least one array of sensing elements is positioned opposite a corresponding point source and where the arrays are arranged in any geometric pattern; and

(b) the at least one array of sensing elements has optical bandpass filters, where the spectral bands passed are different for the sensing arrays either within or between the plurality of reconstruction modules.

50. The system of claim 46 wherein each of the plurality of fixed point sources comprises a circular point source.

51. The system of claim 46 wherein a circle of reconstruction is defined by radially overlapping projection fans from each of the plurality of fixed point sources at the apex and the width of the sensing array at the base.

52. The system of claim 46 wherein the plurality of fixed point sources comprise a point source device selected from the group consisting of:

(a) a pinhole in front of a laser;

(b) an optical fiber;

(c) a short focal length lens in front of a photon source;

(d) an electron beam that irradiates a point on a phosphor surface; and

(e) a high intensity photon source; and

(f) any combination of the above elements (a) through (e).

53. The system of claim 46 wherein the at least one reconstruction cylinder includes_an array of sensing elements selected from the group consisting of charge coupled devices (CCDs), photodiodes, CMOS, CdZnTe, MgI sensors, solid state sensors, a photon-sensitive array of elements in any geometric arrangement, including a linear arrangement.

54. The system of claim 53 wherein the array of sensing elements are centered on a line between the plurality of fixed point sources and the central flow axis.

55. The system of claim 54 wherein the array of sensing elements line up perpendicularly to the central flow axis.

56. The system of claim 46 wherein a volume of reconstruction is defined by radially overlapping projection cones from the point source at the apex and the width of the sensing array at the base.

57. The system of claim 23 wherein offset data is provided for normalizing the system.

58. The system of claim 57 wherein the system is calibrated by i) acquiring images in the absence of any at least one object in the flow tube, and ii) acquiring images of at least one object of known optical properties.

59. The system of claim 58 where the calibration data is reconstructed.

60. The system of claim 58 wherein the at least one object of known optical properties is selected from the group consisting of latex microspheres, polymer microspheres, and oblate spheroids.

61. The system of claim 23 wherein the at least one reconstruction cylinder provides image signals that are reconstructed using filtered backprojection algorithms where the algorithm computes a two dimensional (2D) image of a slice perpendicular to the axis of motion, and the serial stacking of multiple slices generates a three dimensional (3D) image of the at least one object where contrast is a function of the variations in optical density within the at least one object.

62. The system of claim 23 where chromaphors are used to distinguish a number of molecular probes and structural features within a given cell.

63. The system of claim 23 further, wherein the at least one object includes individually stained molecules, comprising serial bandpass filters coupled to the at least one reconstruction cylinder to separate wavelength data and allow the reconstruction and spatial localization of the individually stained molecules.

64. The system of claim 23 wherein the at least one reconstruction cylinder is produced by microfabrication techniques.

65. The system of claim 23 wherein the reconstruction cylinder provides image signals that are reconstructed using filtered backprojection algorithms where the algorithm computes a two dimensional (2D) image of a slice perpendicular to the axis of motion, and the serial stacking of multiple slices generates a three dimensional (3D) image of the at least one object where contrast is a function of variations in probe emission density within the at least one object.

66. A flow optical tomography system for imaging and analysis of microscopic objects, the system comprising:

a pulse height analyzer, a source of photons and a photon sensor, where the source of photons and the photon sensor work together with the pulse height analyzer to operate as a triggering device, where the pulse height analyzer provides a first trigger point for the beginning of a the at least one object, and a second trigger point for the end of the at least one object to create a corresponding trigger signal delivered to a reconstruction cylinder for the purpose of synchronizing object velocity and position with each projection slice.