3D IMAGING OF LIVE CELLS WITH ULTRAVIOLET RADIATION

• US 20120145926A1
• Filed: 02/22/2012
• Published: 06/14/2012
• Est. Priority Date: 02/18/2008
• Status: Active Grant

• Current Assignee: VisionGate, Inc.
• Original Assignee: VisionGate, Inc.
• Inventors: Seibel, Eric J., Fauver, Mark E., Rahn, J. Richard, Nelson, Alan C.
• Granted Patent: US 8,368,035 B2
• Time in Patent Office: Days
• US Class Current: 250/459.1
• CPC Class Codes: G01N 15/1436 the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 2015/1445 Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography
• CPC Class Codes: G01N 2021/1787 Tomographic, i.e. computerised reconstruction from projective measurements
• CPC Class Codes: G01N 21/4795 spatially resolved investigating of object in scattering medium in vivo A61B
• 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 method for 3D imaging of cells in an optical tomography system includes moving a biological object relatively to a microscope objective to present varying angles of view. The biological object is illuminated with radiation having a spectral bandwidth limited to wavelengths between 150 nm and 390 nm. Radiation transmitted through the biological object and the microscope objective is sensed with a camera from a plurality of differing view angles. A plurality of pseudoprojections of the biological object from the sensed radiation is formed and the plurality of pseudoprojections is reconstructed to form a 3D image of the cell.

Claims

1. A method for 3D imaging of a biological object in an optical tomography system comprising:

moving a biological object relative to a microscope objective to present varying angles of view;

illuminating the biological object with radiation having a spectral bandwidth limited to wavelengths between 150 nm and 390 nm;

sensing radiation transmitted through the biological object and the microscope objective with an ultraviolet camera, wherein the sensed radiation comprises at least two selected wavelengths;

forming a plurality of pseudoprojection images of the biological object from the sensed radiation; and

reconstructing the plurality of pseudoprojection images to form a 3D image.

2. The method of claim 1 wherein the spectral bandwidth has wavelengths further limited to between 240 nm and 300 nm.

3. The method of claim 1 wherein the spectral bandwidth has wavelengths further limited to between 260 nm and 265 nm.

4. The method of claim 1 wherein the spectral bandwidth has wavelengths further limited to between 280 nm and 285 nm.

5. The method of claim 1 wherein the biological object is selected from the group consisting of a cell, cell parts, chromosomes, a live cell, a fixed cell, an unfixed cell, a frozen cell, a thawed cell, a desiccated cell, a cloned cell, a cell nucleus, an organelle, a mobile cell, an immobilized cell, deoxyribonucleic acid (DNA), and protein.

6. The method of claim 1 wherein the radiation stimulates native fluorescence from the biological object, further comprising measuring the stimulated fluorescence.

7. The method of claim 1 wherein a size of a voxel in the reconstructed 3D image is known, further comprising measuring a concentration of molecules absorbing the radiation by measuring the absorbance per voxel.

8. The method of claim 1 wherein the biological object is contained in aqueous environment.

9. The method of claim 8 wherein the aqueous environment comprises physiological buffered saline.

10. The method of claim 1 wherein the biological object comprises a live cell.

11. The method of claim 3 wherein the sensed radiation includes imaging signals emanating from DNA.

12. The method of claim 4 wherein the sensed radiation includes imaging signals emanating from protein.

13. The method of claim 1 wherein the sensed radiation includes imaging signals emanating from hydrophilic surfaces.

14. The method of claim 1 wherein illuminating the biological object comprises illuminating with multiple wavelengths.

15. The method of claim 1 wherein forming a plurality of pseudoprojection images of the cell comprises processing images using ratio imaging.

16. The method of claim 15 wherein the ratio imaging includes images formed from wavelengths ranging from 260 nm to 285 nm.

17. The method of claim 1 further comprising harvesting cells.

18. The method of claim 1 wherein the biological object is held in a microcapillary tube.

19. The method of claim 1 wherein the biological object is in a tube configured to recycle cells through an imaging chamber.

20. The method of claim 1 wherein the radiation comprises at least first and second wavelengths selected to enhance natural radiation absorbance by at least one of DNA and protein.

21. The method of claim 1 wherein the radiation comprises at least two selected wavelengths that are pulsed in time series.

22. The method of claim 21 wherein the at least two selected wavelengths that are pulsed in time series emanate from a beamsplitter.

23. The method of claim 1 comprising splitting the radiation transmitted through the biological object into at least two selected wavelengths.

24. The method of claim 23 further comprising sensing the at least two selected wavelengths using a first light detector sensitive to a first wavelength and a second light detector sensitive to a second wavelength.

25. The method of claim 24 wherein splitting radiation comprises directing the radiation through a beamsplitter.

26. The method of claim 25 wherein the beamsplitter is selected from the group consisting of a polarizing beam splitter, a Wollaston prism, a birefringent element, a half-silvered mirror, a 50/50 intensity beamsplitter, a dielectric optically coated mirror, a pellicle film and a dichroic mirrored prism.

27. The method of claim 1 further comprising positioning an adaptive mirror to direct the radiation to the microscope objective.

28. The method of claim 27 further comprising reducing depth-dependent image aberrations by controlling the adaptive mirror with an adaptive optics controller.

29. The method of claim 27 wherein the adaptive mirror is selected from the group consisting of an unpowered adaptive mirror and an adaptive mirror energized at a constant wavefront compensation profile.

30. An optical tomography system for acquiring 3D images comprising:

at least one optical illumination source for producing light having a spectral bandwidth with wavelengths between 150 nm and 390 nm;

an objective lens having a depth of field, the objective lens being located to receive the light;

an axial translation mechanism coupled to translate the objective lens through a scanning range so as to extend the depth of field;

transport means adapted to present a plurality of differing views of a specimen when present in the scanning range;

an ultraviolet sensor for sensing light transmitted through the objective lens;

an image processor coupled to receive data from sensor, wherein the ultraviolet sensor includes

a first light detector sensitive to light having a first wavelength, the first light detector being positioned to sense light passed through the microscope objective; and

a second light detector sensitive to light having a second wavelength, the second light detector being positioned to sense light passed through the microscope objective, where the first light detector and the second light detector are positioned along intersecting optical paths; and

a reconstruction module coupled to the image processor, where the reconstruction module processes the data to form a 3D image of the cell.

31. The system of claim 30 wherein the axial translation mechanism comprises a piezoelectric transducer.

32. The system of claim 30 wherein the at least one radiation source comprises a computer-controlled light source and condenser lens assembly.

33. The system of claim 30 wherein the transport means comprises a microcapillary tube.

34. The system of claim 31 wherein a computer is linked to control the piezoelectric transducer, where the piezoelectric transducer axially moves the objective lens so as to extend the depth of field of the objective lens.

35. The system of claim 30 wherein the spectral bandwidth has wavelengths further limited to between 240 nm and 300 nm.

36. The system of claim 30 wherein the spectral bandwidth has wavelengths further limited to between 260 nm and 265 nm.

37. The system of claim 30 wherein the spectral bandwidth has wavelengths further limited to between 280 nm and 285 nm.

38. The system of claim 30 wherein the biological object is selected from the group consisting of a cell, cell parts, chromosomes, a live cell, a fixed cell, an unfixed cell, a frozen cell, a thawed cell, a desiccated cell, a cloned cell, a cell nucleus, an organelle, a mobile cell, an immobilized cell, DNA, and protein.

39. The system of claim 30, wherein the light stimulates native fluorescence from the biological object.

40. The system of claim 30, wherein a size of a voxel in the reconstructed 3D image is known, further comprising means for measuring a concentration of molecules absorbing the radiation by measuring the absorbance per voxel.

41. The system of claim 30 wherein the transport means contains a specimen including a biological object in an aqueous environment.

42. The system of claim 41 wherein the aqueous environment comprises physiological buffered saline.

43. The system of claim 41 wherein the biological object comprises a live cell.

44. The system of claim 30 wherein the sensed radiation includes imaging signals emanating from DNA.

45. The system of claim 30 wherein the sensed light includes imaging signals emanating from protein.

46. The method of claim 30 wherein the sensed light includes imaging signals emanating from hydrophilic surfaces.

47. The system of claim 30 wherein the at least one optical illumination source generates light having multiple wavelengths.

48. The system of claim 30 wherein the reconstruction module includes a ratio imaging process.

49. The system of claim 47 wherein the ratio imaging process includes images formed from wavelengths ranging from 260 nm to 280 nm.

50. The system of claim 30 wherein the transport means comprises a straight tube of more than one channel.

51. The system of claim 30 wherein the ultraviolet sensor is an ultraviolet pixel array detector.

52. The system of claim 30 wherein the spectral bandwidth is limited to wavelengths selected to enhance natural radiation absorbance by protein.

53. The system of claim 30 wherein the at least one optical illumination source comprises multiple sources transmitting at least two selected wavelengths.

54. The system of claim 30 wherein the transport means is adapted to hold a specimen including a biological object selected from the group consisting of cells, cell parts, chromosomes, a live cell, a fixed cell, an unfixed cell, a frozen cell, a thawed cell, a desiccated cell, a cloned cell, a cell nucleus, an organelle, DNA, and protein.

55. The system of claim 30 wherein the at least one of the first and second wavelengths is selected to enhance natural radiation absorbance by DNA.

56. The system of claim 55 further comprising a beamsplitter positioned to split radiation transmitted through the biological object into at least two selected wavelengths that are separately transmitted to the first light detector and the second light detector.

57. The system of claim 55 further wherein the first light detector comprises a CCD sensor.

58. The system of claim 55 wherein the first light detector is sensitive to radiation having a wavelength matching the natural absorbance of human DNA.

59. The system of claim 55 wherein the second light detector is sensitive to radiation having a wavelength matching the natural absorbance of protein.

60. The system of claim 55 wherein the first light detector is sensitive to radiation having a wavelength that includes imaging signals emanating from hydrophilic surfaces.

61. The system of claim 56 wherein the beamsplitter is selected from the group consisting of a polarizing beam splitter, a Wollaston prism, a birefringent element, a half-silvered mirror, a 50/50 intensity beamsplitter, a dielectric optically coated mirror, a pellicle film and a dichroic mirrored prism.

62. The system of claim 30 further comprising at least one filter interposed between the ultraviolet sensor and the transmitted radiation.

63. The system of claim 30 wherein the at least one radiation source comprises a set of UV light sources generating different wavelengths.