Apparatus and method for dynamic cellular probing and diagnostics using holographic optical forcing array

• US 20090128825A1
• Filed: 10/30/2008
• Published: 05/21/2009
• Est. Priority Date: 10/17/2005
• Status: Active Grant

• Current Assignee: Arryx, Inc. (Haemonetics Corporation)
• Original Assignee: Arryx, Inc. (Haemonetics Corporation)
• Inventors: Akcakir, Osman
• Granted Patent: US 8,149,416 B2
• Time in Patent Office: Days
• US Class Current: 356/457
• CPC Class Codes: G01B 9/021 using holographic techniques
• CPC Class Codes: G01N 21/453 Holographic interferometry for dimensional measurements G01B9/021 - G01B9/029
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01Q 20/02 by optical means
• CPC Class Codes: G01Q 30/04 Display or data processing devices
• CPC Class Codes: G01Q 60/20 Fluorescence
• CPC Class Codes: G03H 1/0443 Digital holography, i.e. recording holograms with digital recording means holobject computation G03H1/0866
• CPC Class Codes: G03H 2001/0033 in hologrammetry for measuring or analysing
• CPC Class Codes: G03H 2001/0077 for optical manipulation, e.g. holographic optical tweezers [HOT]
• CPC Class Codes: G03H 2001/045 Fourier or lensless Fourier arrangement
• CPC Class Codes: G03H 2001/0458 Temporal or spatial phase shifting, e.g. parallel phase shifting method
• CPC Class Codes: G03H 2223/23 Diffractive element

Abstract

The present invention utilizes a holographic optical forcing array for dynamic cellular probing and diagnostics. A holographic optical trapping system generates optical forces on objects so that deformations thereof may be quantified. In one embodiment, digital holography is used to generate an interference pattern, and an analysis thereof determines the phase profile which yields a measurement of the objects'"'"' shape deformation using only one image. In another embodiment, phase-stepped holography allows the phase profile of an object to be measured using only one image, by using a holographic optical element to make phase-shifted replicas of the beam in space. In another embodiment, the optical forcing array applies optical forces to beads placed on the objects'"'"' surface, deforming the objects. The beads'"'"' position is determined by applying Mie theory, and analysis thereof yields the three dimensional position of the beads, and a measurement of the deformation displacement on the objects'"'"' surface.

Claims

1. An apparatus for detecting deformation in a sample contained in a sample chamber, said sample containing at least one object, comprising:

a light source which emits a first light beam which illuminates at least one object;

an optical forcing mechanism which provides an optically generated force on at least one object, said optical forcing mechanism which includes;

a laser which emits a second light beam; and

a spatial light modulator which diffracts said second light beam and which applies the optically generated force to the object; and

a detector which detects an interference pattern between a first portion of said first light beam which is undiffracted, and a second portion of said first light beam which is diffracted by the at least one object in the sample, to determine a quantitative deformation of the at least one object.

2. The apparatus according to claim 1, wherein said optical forcing mechanism further comprises:

a microscope, including an objective lens.

3. The apparatus according to claim 1, wherein said light source is an imaging laser or a superluminescent diode.

4. The apparatus according to claim 1, further comprising:

a computer having a program which analyzes said interference pattern.

5. The apparatus according to claim 2, wherein said optical forcing mechanism modifies said second light beam from said spatial light modulator to diffract said second light beam into multiple diffracted beamlets, and where said diffracted beamlets are applied to said at least one object to impart forces onto the at least one object.

6. The apparatus according to claim 5, wherein said diffracted beamlets act as optical force probes which apply pressure to the at least one object in the sample simultaneously.

7. The apparatus according to claim 6, further comprising:

a first dichroic mirror, which couples the diffracted second light beam from said spatial light modulator, into said objective lens; and

a second dichroic mirror, which directs said diffracted and undiffracted light beams from said first light source to said detector after having passed through the sample chamber.

8. The apparatus according to claim 7, wherein said first portion of said first light beam which is undiffracted, is transmitted through the sample containing the at least one object without being diffracted by said at least one object, to yield an undiffracted reference beam which later recombines and interferes with said second portion of said first light beam that is diffracted by the at least one object, yielding said interference pattern.

9. The apparatus according to claim 8, further comprising:

at least one bead, said bead which is placed on a surface of the at least one object, and to which said optical forcing mechanism applies said optically generated force;

wherein said optically generated force on said at least one bead exerts pressure on the at least one object underlying said at least one bead to deform the at least one object.

10. The apparatus according to claim 9, wherein a three-dimensional position of each said at least one bead is determined by applying Mie theory to an in-line holographic image of said bead, which is an interference pattern between a diffracted beam and an undiffracted beam.

11. The apparatus according to claim 10, wherein a measurement of deformation displacement on said surface of said at least one object is performed by determining said position of said bead before and after an application of said optically generated force.

12. The apparatus according to claim 8, further comprising:

a mirror which directs said undiffracted and diffracted light beams from said first light source, via said second dichroic mirror, to said detector.

13. The apparatus according to claim 7, further comprising:

a first beam splitter which splits said first light beam into a reference beam and an object beam, said object beam which illuminates said at least one object, and said reference beam which is undiffracted and directed to said detector;

wherein said object beam and said reference beam are combined in an off-axis geometry at said detector; and

wherein said detector detects said interference pattern between said reference beam and said object beam.

14. The apparatus according to claim 13, wherein said second dichroic mirror directs said object beam to said detector.

15. The apparatus according to claim 14, further comprising:

a second beam splitter which directs said reference beam from said first beam splitter, and said object beam from said second dichroic mirror, to said detector.

16. The apparatus according to claim 15, wherein at said detector, said reference beam is temporally coherent with said object beam, such that said interference pattern is generated at said detector;

and

wherein a quantitative phase profile of said at least one object is obtained from said interference pattern.

17. The apparatus according to claim 15, further comprising:

a holographic optical element disposed between said second dichroic mirror and said second beam splitter, which diffracts said object beam into four beamlets and directs said beamlets to said second beam splitter and to said detector, generating four spatially phase-stepped replicas of said object beam.

18. The apparatus according to claim 17, wherein said holographic optical element makes phase-stepped replicas of images of said at least one object which are diffracted into four quadrants of said detector and simultaneously interfered with said reference beam.

19. The apparatus according to claim 18, further comprising:

a computer having a program which computes a phase profile of said at least one object from said four holographically phase-stepped replica images interfered with said reference beam.

20. The apparatus according to claim 1, wherein said detector is a high-speed detector camera.

21. The apparatus according to claim 1, wherein said forcing laser is a continuous wave or pulsed forcing laser.

22. The apparatus according to claim 2, wherein said at least one object is an adherent cell.

23. The apparatus according to claim 22, wherein said at least one cell is grown on an amorphous fluoropolymer-coated cover glass.

24. The apparatus according to claim 22, wherein said at least one cell is grown on a cover glass coated with protein.

25. The apparatus according to claim 17, further comprising:

a transfer lens disposed between said holographic optical element and said second beam splitter.

26. The apparatus according to claim 17, wherein a measurement of a phase distribution before and after optical forcing of said at least one object, provides an optical path-length shift determination which denotes an optical deformability of said at least one object.

27. The apparatus according to claim 2, further comprising:

a movable sample stage which moves said sample to a location of said second light beam of said optical forcing mechanism.

28. The apparatus according to claim 2, further comprising:

a mechanism which moves said second light beam of said optical forcing mechanism, said mechanism including movable mirrors and acousto-optic modulators.

29. An endoscope, comprising:

a light source which emits a first light beam which illuminates at least one object;

an optical forcing mechanism which provides an optically generated force on the at least one object, said optical forcing mechanism which includes;

a laser which emits a second light beam; and

a spatial light modulator which diffracts said second light beam and which applies the optically generated force to the object; and

a detector which detects an interference pattern between a first portion of said first light beam which is undiffracted, and a second portion of said first light beam which is diffracted by the at least one object, to determine a quantitative deformation of the at least one object.

30. An apparatus for detecting deformation in a sample contained in a sample chamber, said sample containing at least one object, comprising:

a light source which emits a first light beam which illuminates the at least one object;

an optical forcing mechanism which provides an optically generated force on the at least one object, said optical forcing mechanism which includes;

a laser which emits a second light beam which applies the optically generated force to the object; and

a detector which detects an interference pattern between a first portion of said first light beam which is undiffracted, and a second portion of said first light beam which is diffracted by the at least one object in the sample, to determine a quantitative deformation of the at least one object.

31. A method of detecting deformation in at least one object of a sample, comprising:

illuminating said at least one object on a substrate using a first light beam generated by a light source;

applying an optically generated force on said at least one object using a second light beam generated by a holographic optical trapping mechanism;

measuring an interference pattern between a first portion of said first light beam which is undiffracted, and a second portion of said first light beam which is diffracted by the at least one object in the sample, to determine a quantitative deformation of the at least one object.

32. The method according to claim 31, wherein said applying step comprises:

diffracting said second light beam into multiple diffracted beamlets using a spatial light modulator which modifies said second light beam; and

applying said diffracted beamlets to said at least one object to impart forces onto said at least one object.

33. The method according to claim 31, wherein said substrate is included in a microscope, said microscope having an objective lens.

34. The method according to claim 31, wherein said light source is an imaging laser or a superluminescent diode.

35. The method according to claim 31, further comprising:

a computer having a program which analyzes said interference pattern.

36. The method according to claim 32, wherein said diffracted beamlets act as optical force probes which apply pressure to the at least one object in the sample simultaneously.

37. The method according to claim 32, further comprising:

coupling said diffracted second light beam from said spatial light modulator into said objective lens using a first dichroic mirror; and

directing said diffracted and undiffracted light beams from said first light source to said detector after having passed through the sample chamber using a second dichroic mirror.

38. The method according to claim 32, further comprising:

transmitting said first portion of said first light beam which is undiffracted, through the sample containing said at least one object without being diffracted by said at least one object; and

yielding an undiffracted reference beam which later recombines and interferes with said second portion of said first light beam that is diffracted by at least one object, to yield said interference pattern;

39. The method according to claim 32, further comprising:

disposing at least one bead on a surface of the at least one object; and

applying said optically generated force to said at least one bead, such that optical forces are applied to said bead;

wherein said optically generated force on said at least one bead exerts pressure on the at least one object underlying said at least one bead to deform the at least one object.

40. The method according to claim 39, further comprising:

determining a three dimensional position of each of said at least one bead by applying Mie theory to an in-line holographic image of said bead, which is an interference pattern between a diffracted beam and an undiffracted beam.

41. The method according to claim 40, further comprising:

determining said position of said bead before and after an application of said optically generated force to measure a deformation displacement on said surface of said at least one object.

42. The method according to claim 41, further comprising:

directing said undiffracted and diffracted light beams from said first light source using a mirror, to said detector via said second dichroic mirror.

43. The method according to claim 38, further comprising:

splitting said first light beam into a reference beam and an object beam using a first beam splitter, said object beam which illuminates said at least one object, and said reference beam which is undiffracted and directed to said detector;

combining said object beam and said reference beam in an off-axis geometry at said detector; and

detecting said interference pattern between said reference beam and said object beam.

44. The method according to claim 43, further comprising:

directing said object beam to said detector using said second dichroic mirror.

45. The method according to claim 44, further comprising:

directing said reference beam from said first beam splitter to said detector using a second beam splitter; and

directing said object beam from said second dichroic mirror to said detector using said second beam splitter.

46. The method according to claim 45, wherein at said detector, said reference beam is temporally coherent with said object beam, such that said interference pattern is generated at said detector;

and

wherein a quantitative phase profile of said at least one object is obtained from said interference pattern.

47. The method according to claim 44, further comprising:

disposing a holographic optical element between said second dichroic mirror and said second beam splitter; and

diffracting said object beam into four beamlets and directing said beamlets to said second beam splitter and to said detector, generating four spatially phase-stepped replicas of said object beam.

48. The method according to claim 47, further comprising:

making phase-stepped replicas of images of said at least one object using said holographic optical element, which are diffracted into four quadrants of said detector and simultaneously interfered with said reference beam.

49. The method according to claim 48, further comprising:

computing a phase profile of said at least one object from said four holographically phase-stepped replica images interfered with said reference beam.

50. The method according to claim 31, wherein said detector is a fast-detector camera.

51. The method according to claim 31, wherein said laser is a continuous wave or pulsed forcing laser.

52. The method according to claim 31, wherein said at least one object is an adherent cell.

53. The method according to claim 52, wherein said at least one cell is grown on an amorphous fluoropolymer-coated cover glass.

54. The method according to claim 52, wherein said at least one cell is grown on a cover glass coated with protein.

55. The method according to claim 48, further comprising:

disposing a transfer lens between said holographic optical element and said second beam splitter.

56. The method according to claim 31, wherein a measurement of a phase distribution before and after optical forcing of said at least one object, provides an optical path-length shift determination which denotes an optical deformability of said at least one object.

57. The method according to claim 31, wherein a movable sample stage moves said sample to a location of said second light beam of said optical forcing mechanism.

58. The method according to claim 31, further comprising:

moving said second light beam of said optical forcing mechanism to said at least one object, said mechanism including movable mirrors and acousto-optic modulators.