Tomographic phase microscopy

• US 20090125242A1
• Filed: 07/10/2008
• Published: 05/14/2009
• Est. Priority Date: 07/10/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Fang-Yen, Christopher M., Choi, Wonshik, Dasari, Ramachandra Rao, Feld, Michael S.
• Granted Patent: US 8,848,199 B2
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: G01N 2021/458 using interferential sensor, e.g. sensor fibre, possibly on optical waveguide
• CPC Class Codes: G01N 21/45 using interferometric methods; using Schlieren methods
• CPC Class Codes: G01N 21/51 inside a container, e.g. in an ampoule G01N21/53 takes precedence
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/061 Sources
• CPC Class Codes: G01N 2201/105 Purely optical scan
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing
• CPC Class Codes: G03H 1/0443 Digital holography, i.e. recording holograms with digital recording means holobject computation G03H1/0866
• CPC Class Codes: G03H 1/265 Angle multiplexing; Multichannel holograms G03H1/268 takes precedence
• CPC Class Codes: G03H 2001/0458 Temporal or spatial phase shifting, e.g. parallel phase shifting method

Abstract

The present invention relates to systems and methods for quantitative three-dimensional mapping of refractive index in living or non-living cells, tissues, or organisms using a phase-shifting laser interferometric microscope with variable illumination angle. A preferred embodiment provides tomographic imaging of cells and multicellular organisms, and time-dependent changes in cell structure and the quantitative characterization of specimen-induced aberrations in high-resolution microscopy with multiple applications in tissue light scattering.

Claims

1. An interferometric method for measuring refractive index of a medium comprising:

scanning light along a first scanning light path through a medium at a plurality of angles;

combining the light transmitted through the medium with a reference light;

detecting the combined light; and

determining a distribution of a refractive index of the medium.

2. The method of claim 1 further comprising positioning a sample in the medium to be measured relative to the first scanning light path.

3. The method of claim 2 wherein the sample is a biological sample.

4. The method of claim 2 wherein an index of refraction of the sample is altered by less than 5%.

5. The method of claim 4 wherein an index of refraction of the sample is altered by less than 1%.

6. The method of claim 5 wherein an index of refraction of the sample is not altered.

7. The method of claim 3 further comprising adding a substance to the medium or to the biological sample prior to said step of determining, wherein adding the substance alters an index of refraction of the biological sample by less than 5%.

8. The method of claim 7 wherein adding the substance alters an index of refraction of the biological sample by less than 1%.

9. The method of claim 3 further comprising adding a substance to the medium or to the biological sample prior to said step of determining, wherein adding the substance alters an index of refraction of the biological sample.

10. The method of claim 3 wherein the biological sample is a cell, a plurality of cells, a tissue, or an organism.

11. The method of claim 3 wherein the medium is a culture medium.

12. The method of claim 2 further comprising changing the medium while the sample remains in place.

13. The method of claim 12 wherein an index of refraction of the sample is not altered.

14. The method of claim 13 wherein an index of refraction of the sample is altered.

15. The method of claim 3 wherein an index of refraction in a single cell is measured.

16. The method of claim 3 wherein an autofluorescence in a single cell is measured.

17. The method of claim 3 wherein a scattering spectrum in a single cell is measured.

18. The method of claim 3 wherein a size or volume is measured for the biological sample or a portion thereof.

19. The method of claim 3 wherein a cell size distribution is measured for the biological sample or a portion thereof.

20. The method of claim 3 wherein a characteristic of a nucleic acid is measured in the biological sample or a portion thereof.

21. The method of claim 1 wherein the plurality of angles are in the range from +60 to −

60 degrees.

22. The method of claim 21 further comprising performing a constrained iteration process to supply missing-angle projections.

23. The method of claim 2 further comprising obtaining a plurality of images of the sample at a rate of at least four frames per second.

24. The method of claim 1 further comprising determining a distribution of fluorescence in the medium.

25. The method of claim 24 wherein the medium comprises a biological sample, and the fluorescence is autofluorescence of the sample or is caused by a fluorescent label added to the sample.

26. The method of claim 1 further comprising holding a tissue sample stationary relative to the scanning light.

27. The method of claim 1 further comprising detecting a plurality of phase images at different angles through the medium.

28. The method of claim 1 further comprising modulating a frequency of the reference light.

29. The method of claim 1 further comprising detecting a light scattering spectrum of the medium.

30. The method of claim 1 further comprising moving a focal plane of the light through the medium.

31. The method of claim 1 further comprising determining a three-dimensional distribution of the refractive index in a portion of the medium.

32. The method of claim 1 further comprising illuminating the medium with a broadband light source.

33. The method of claim 1 further comprising illuminating the medium with a coherent light source to induce autofluorescence in the medium.

34. The method of claim 1 further comprising measuring a refractive index distribution in a live organism.

35. The method of claim 1 further comprising splitting light from a light source to provide the scanning light and the reference light.

36. The method of claim 1 further comprising using an actuator to scan the scanning light through the sample at a plurality of angles.

37. The method of claim 36 further comprising recording an image of the combined light at each of the plurality of angles.

38. The method of claim 37 wherein a phase shift between the light transmitted through the medium and the reference light between successive recorded images is π

/2.

39. The method of claim 1 wherein the reference light is directed through an acousto-optic modulator.

40. The method of claim 36 wherein the actuator drives a tilting mirror to deflect the scanning light at different angles.

41. The method of claim 1 further comprising a lens to focus the scanning light at a back focal plane of a condenser lens.

42. The method of claim 1 further comprising measuring a first electric field image and a first quantitative phase image at a first focal location in the medium and subsequently calculating a second electric field image and a second quantitative phase image at a selected distance from the first focal location.

43. The method of claim 42 wherein the step of calculating comprises determining a Fourier transform and applying a filter to obtain the second electric field image and the second quantitative phase image at each of the plurality of angles.

44. A method of diagnosing a disease or medical condition comprising performing the method of claim 1 wherein the medium comprises a cell or tissue sample from a subject suspected of having said disease or medical condition.

45. The method of claim 44 wherein the subject is suspected of having cancer.

46. The method of claim 45 wherein the subject is suspected of having cervical or uterine cancer.

47. The method of claim 46 wherein the cell or tissue sample is positioned in the medium to be measured relative to the first scanning light path, and a change in a refractive index distribution in the sample or a portion thereof is determined after introduction of acetic acid into the medium.

48. A tomography system for measuring a medium comprising:

a first light source;

a first light path coupling light from the light source onto the medium;

a scanner that scans the first light path through the medium at different angles;

a second light path that couples a reference light onto light transmitted through the medium;

a detector that detects the reference light combined with the light transmitted through the medium; and

a processor that determines a three-dimensional distribution of a refractive index of the medium from the detected light.

49. The system of claim 48 further comprising a frequency modulator that modulates a frequency of the reference light.

50. The system of claim 49 wherein the frequency modulator is an acousto-optic modulator.

51. The system of claim 49 further comprising a beam expander in the second light path downstream from the frequency modulator.

52. The system of claim 48 comprising a microscope.

53. The system of claim 48 further comprising a second light source for broadband illumination or fluorescence excitation.

54. The system of claim 53 comprising a second light source for fluorescence excitation and further comprising a second detector that detects fluorescence emission.

55. The system of claim 48 wherein the first light source is capable of emitting ultraviolet light.

56. The system of claim 48 wherein the first light source is capable of emitting light at 3 or more distinct wavelengths.

57. The system of claim 48 wherein a detected image is processed with the three-dimensional distribution of the refractive index to provide a light scattering spectrum.

58. The system of claim 57 wherein a detector is positioned in an image plane of the medium to detect an image that is Fourier transformed to provide a light scattering spectrum.

59. The system of claim 48 wherein the scanner further comprises a plurality of positions to move a focal plane of light through the medium.

60. The system of claim 48 wherein the scanner further comprises an actuator and a scanning optical element mounted on the actuator, wherein the actuator moves the scanning optical element to provide the different angles of the first light path through the medium.

61. A spatial fringe pattern demodulation method for performing tomographic phase microscopy, the method comprising:

scanning light along a first light path through a medium at a plurality of angles;

combining the light transmitted through the medium with a reference light;

detecting the combined light; and

determining a phase image of the medium using spatial fringe pattern demodulation.

62. The method of claim 61, further comprising positioning a sample in the medium to be measured relative to the first scanning light path.

63. The method of claim 62, wherein the sample is a biological sample.

64. The method of claim 62, wherein an index of refraction of the sample is altered by less than 5%.

65. The method of claim 64, wherein an index of refraction of the sample is altered by less than 1%.

66. The method of claim 65, wherein an index of refraction of the sample is not altered.

67. The method of claim 63, further comprising adding a substance to the medium or to the biological sample prior to said step of determining, wherein adding the substance alters an index of refraction of the biological sample by less than 5%.

68. The method of claim 67, wherein adding the substance alters an index of refraction of the biological sample by less than 1%.

69. The method of claim 63, further comprising adding a substance to the medium or to the biological sample prior to said step of determining, wherein adding the substance alters an index of refraction of the biological sample.

70. The method of claim 63, wherein the biological sample is a cell, a plurality of cells, a tissue, or an organism.

71. The method of claim 63, wherein the medium is a culture medium.

72. The method of claim 62, further comprising changing the medium while the sample remains in place.

73. The method of claim 72, wherein an index of refraction of the sample is not altered.

74. The method of claim 72, wherein an index of refraction of the sample is altered.

75. The method of claim 63, wherein phase image of a single cell is measured.

76. The method of claim 63, wherein an autofluorescence in a single cell is measured.

77. The method of claim 63, wherein a scattering spectrum in a single cell is measured.

78. The method of claim 63, wherein a size or volume is measured for the biological sample or a portion thereof.

79. The method of claim 63, wherein a cell size distribution is measured for the biological sample or a portion thereof.

80. The method of claim 63, wherein a characteristic of a nucleic acid is measured in the biological sample or a portion thereof.

81. The method of claim 61, wherein the plurality of angles are in the range from +60 to −

60 degrees.

82. The method of claim 81, further comprising performing a constrained iteration process to supply missing-angle projections.

83. The method of claim 61, further comprising obtaining a plurality of images of the sample at a rate of at least four frames per second.

84. The method of claim 83, wherein images of the sample are obtained at a rate of at least 30 frames per second.

85. The method of claim 61, further comprising determining a distribution of fluorescence in the medium.

86. The method of claim 85, wherein the medium comprises a biological sample, and the fluorescence is autofluorescence of the sample or is caused by a fluorescent label added to the sample.

87. The method of claim 61 wherein the transmitted light comprises ultraviolet light.

88. The method of claim 61 wherein a plurality of different wavelengths of light are transmitted.

89. A method of diagnosing a disease or medical condition comprising performing the method of claim 61, wherein the medium comprises a cell or tissue sample from a subject suspected of having said disease or medical condition.

90. The method of claim 89, wherein the subject is suspected of having cancer.

91. The method of claim 90, wherein the subject is suspected of having cervical or uterine cancer.

92. The method of claim 91, wherein the cell or tissue sample is positioned in the medium to be measured relative to the first scanning light path, and a change in a refractive index distribution in the sample or a portion thereof is determined after introduction of acetic acid into the medium.

93. A tomography system for measuring a medium comprising:

a light source;

a first light path coupling light from the light source onto the medium;

a scanner that scans the first light path through the medium at different angles;

a second light path that couples a reference light onto light transmitted through the medium;

a detector that detects the reference light combined with the light transmitted through the medium; and

a processor that determines a phase image of the medium from the detected light using spatial fringe pattern demodulation.

94. The tomography system of claim 93 wherein said reference light is enlarged by a beam expander prior to coupling with the light transmitted through the medium.

95. The tomography system of claim 93 that is capable of determining said phase image of the medium repeatedly at a rate of at least 30 times per second.

96. The system of claim 93 comprising a microscope.

97. The system of claim 93 further comprising a second light source for broadband illumination or fluorescence excitation.

98. The system of claim 97 comprising a second light source for fluorescence excitation and further comprising a second detector that detects fluorescence emission.

99. The system of claim 93 wherein the first light source is capable of emitting ultraviolet light.

100. The system of claim 93 wherein the first light source is capable of emitting light at 3 or more distinct wavelengths.

101. The system of claim 93 wherein the scanner further comprises an actuator and a scanning optical element mounted on the actuator, wherein the actuator moves the scanning optical element to provide the different angles of the first light path through the medium.

102. A synthetic aperture method for measuring refractive index of a medium comprising:

transmitting a focused beam of light along a first light path through a medium;

combining the light transmitted through the medium with a reference light;

detecting the combined light;

determining a phase image and an amplitude image of the medium;

combining the phase and amplitude images to form an electrical field image of the medium; and

determining the distribution of a refractive index of the medium using synthetic aperture analysis.

103. The method of claim 102 wherein the medium is in translational motion and a series of refractive index distributions of the medium are formed.

104. The method of claim 102, further comprising positioning a sample in the medium to be measured relative to the first scanning light path.

105. The method of claim 104, wherein the sample is in translational motion and a series of refractive index distributions of the sample are formed.

106. The method of claim 104, wherein the sample is a biological sample.

107. The method of claim 104, wherein an index of refraction of the sample is altered by less than 5%.

108. The method of claim 107, wherein an index of refraction of the sample is altered by less than 1%.

109. The method of claim 108, wherein an index of refraction of the sample is not altered.

110. The method of claim 106, further comprising adding a substance to the medium or to the biological sample prior to said step of determining, wherein adding the substance alters an index of refraction of the biological sample by less than 5%.

111. The method of claim 110, wherein adding the substance alters an index of refraction of the biological sample by less than 1%.

112. The method of claim 106, further comprising adding a substance to the medium or to the biological sample prior to said step of determining, wherein adding the substance alters an index of refraction of the biological sample.

113. The method of claim 106, wherein the biological sample is a cell, a plurality of cells, a tissue, or an organism.

114. The method of claim 106, wherein the biological sample comprises two or more distinct cell types.

115. The method of claim 106, wherein the medium is a culture medium.

116. The method of claim 104, further comprising flowing the medium and the sample across the first light path.

117. The method of claim 106, wherein an index of refraction in a single cell is measured.

118. The method of claim 106, wherein an autofluorescence in a single cell is measured.

119. The method of claim 106, wherein a scattering spectrum in a single cell is measured.

120. The method of claim 106, wherein a size or volume is measured for the biological sample or a portion thereof.

121. The method of claim 106, wherein a cell size distribution is measured for the biological sample or a portion thereof.

122. The method of claim 106, wherein a characteristic of a nucleic acid is measured in the biological sample or a portion thereof.

123. The method of claim 102, further comprising obtaining a plurality of images of the sample at a rate of at least four frames per second.

124. The method of claim 102, further comprising determining a distribution of fluorescence in the medium.

125. The method of claim 124, wherein the medium comprises a biological sample, and the fluorescence is autofluorescence of the sample or is caused by a fluorescent label added to the sample.

126. The method of claim 102, further comprising modulating a frequency of the reference light.

127. A method of diagnosing a disease or medical condition comprising performing the method of claim 102, wherein the medium comprises a cell or tissue sample from a subject suspected of having said disease or medical condition.

128. The method of claim 127, wherein the subject is suspected of having cancer.

129. The method of claim 128, wherein the subject is suspected of having cervical or uterine cancer.

130. The method of claim 129, wherein the cell or tissue sample is positioned in the medium to be measured relative to the first scanning light path, and a change in a refractive index distribution in the sample is determined after introduction of acetic acid into the medium.

131. A method of performing flow cytometry, comprising carrying out the method of claim 102, wherein a series of refractive index distributions are determined for a plurality of cells in translational motion.

132. A tomography system for measuring a translocating medium comprising:

a first light source;

a first light path coupling focused light from the light source onto the medium;

a second light path that couples a reference light onto light transmitted through the medium;

a detector that detects the reference light combined with the light transmitted through the medium; and

a processor that determines a refractive index distribution of the medium from the detected light using synthetic aperture analysis.

133. The tomography system of claim 132 further comprising a cylindrical lens for focusing light onto the medium.

134. The tomography system of claim 132 further comprising a cylindrical lens for expanding in focal axis the light transmitted through the medium.

135. The tomography system of claim 132 further comprising a frequency modulator that modulates a frequency of the reference light.

136. The system of claim 135 wherein the frequency modulator is an acousto-optic modulator.

137. The system of claim 135 further comprising a beam expander in the second light path downstream from the frequency modulator.

138. The system of claim 132 comprising a microscope.

139. The system of claim 132 further comprising a second light source for broadband illumination or fluorescence excitation.

140. The system of claim 139 comprising a second light source for fluorescence excitation and further comprising a second detector that detects fluorescence emission.