Analytical system and method for analyzing nonlinear optical signals

• US 7,545,494 B2
• Filed: 07/01/2003
• Issued: 06/09/2009
• Est. Priority Date: 07/23/2003
• Status: Expired due to Fees

• Current Assignee: Laser-Laboratorium Gottingen
• Original Assignee: Bayer Technology Services GmbH (Bayer AG)
• Inventors: Balet, Laurent P., Marowsky, Gerd, Haiml, Markus, Duveneck, Gert L.
• Primary Examiner(s): EVANS, FANNIE L
• Application Number:
  US10/565,909
• Publication Number:
  US 20060291772A1
• Time in Patent Office: 2,170 Days
• Field of Search: 356/317, 356/318, 250/458.1, 250/459.1, 250/461.1, 250/461
• US Class Current: 356/317
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Abstract

A method for the ultrasensitive simultaneous measurement of nonlinear optical emission signals in one or two local dimensions wherein excitation light is irradiated in modulated form from at least one light source into an interactive space in which one or several emissions that are nonlinearly correlated with the excitement light can be excited. The light emanating from the interactive spaces is measured using a one or two-dimensional detector array. Measured data is then transmitted to a computer and formatted in a one or two-dimensional data matrix. Further, non-correlated portions of the light emanating from the interactive spaces that are linearly proportionate to the intensity of the excitement light available in the interactive spaces are separated from portions of the light emanating from the interactive spaces which are not linearly proportionate. The invention also relates to an analytical system for carrying out this method.

Claims

1. A method for highly sensitive simultaneous measurement of nonlinear optical emission signals, spatially resolved in one or two spatial dimensions, comprising:

radiating excitation light from at least one light source in a power-modulated and/or pulse-duration-modulated form into interaction spaces, in each of which one or a plurality of emissions that are correlated nonlinearly with the excitation light can be excited,

measuring light emerging from said interaction spaces by means of a one- or two-dimensional detector array,

transmitting measurement data from said detector array to a computer and formatting the data in a one- or multidimensional data matrix,

characterized in that data representative of those portions of the light emerging from the interaction spaces which are linearly proportional to the intensity of the excitation light available in the interaction spaces are separated from data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the available excitation light intensity.

2. The method as claimed in claim 1, wherein the method does not comprise any spectral filtering of the light that is to be detected and emerge from the interaction spaces.

3. The method as claimed in claim 1, wherein the method is carried out in combination with a spectral filtering of the light that is to be detected and emerge from the interaction spaces.

4. The method as claimed in claim 1, wherein said one- or two-dimensional detector array is selected from the group consisting of CCD cameras, CCD chips, CMOS cameras, CMOS chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers, wherein a phase-sensitive demodulation is capable of being integrated into said detector array.

5. The method as claimed in claim 1, wherein the modulation of the excitation light radiated into an interaction space is effected by means of optomechanical and/or acousto-optical and/or electro-optically active auxiliary means.

6. The method as claimed in claim 5, wherein said optomechanical and/or acousto-optical and/or electro-optically active auxiliary means are selected from the group consisting of mechanical shutters and rotating choppers which in each case alternately block and release the light path between the excitation light source and the interaction space, polarization-selective components that are locally or temporally variable in terms of their transmission, acousto-optical modulators and modulators based on interference effects.

7. The method as claimed in one of claims 1, wherein the modulation of the excitation light radiated into an interaction space is effected by means of direct, active modulation of the light radiated from the excitation light source.

8. The method as claimed in claim 7, wherein the modulation of the excitation light radiated into an interaction space is effected by means of modulation of the excitation current of a semiconductor laser as excitation light source.

9. The method as claimed in claim 1, wherein the modulation of the excitation light radiated into an interaction space is effected periodically.

10. The method as claimed in claim 1, wherein the modulation of the excitation light radiated into an interaction space is effected non-periodically.

11. The method as claimed in claim 1, wherein the modulation of the excitation light radiated into an interaction space consists of modulation of the intensity radiated in.

12. The method as claimed in claim 1, wherein the modulation of the excitation light radiated into an interaction space consists of the simultaneous modulation of the pulse duration and the peak power of the excitation light radiated in.

13. The method as claimed in claim 1, wherein the method is effected without detection of the modulated excitation light or a measurement variable proportional thereto.

14. The method as claimed in claim 1, wherein, in addition to the detection of the light emerging from the interaction spaces, the detection of the modulated excitation light or a measurement variable proportional thereto is detected.

15. The method as claimed in claim 1, wherein the detection of the light emerging from the interaction spaces is effected temporally correlated with the modulation of the excitation light power.

16. The method as claimed in claim 15, wherein the detection of the light emerging from the interaction spaces is effected with a frequency corresponding to an integer multiple of the modulation frequency of the excitation light power.

18. The method as claimed in claim 1, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a parallel Taylor expansion.

19. The method as claimed in claim 1, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a harmonic analysis.

20. The method as claimed in claim 1, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected by means of a stepped modulation of the excitation light power.

21. The method as claimed in claim 1, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a four-step algorithm for the modulation of the excitation light power.

23. The method as claimed in claim 21, wherein the data representative of the light emerging from the interaction spaces obtained using a four-step algorithm for the modulation are multiplied by correction factors.

24. The method as claimed in claim 23, wherein the correction factors are determined from measured excitation light powers.

25. The method as claimed in claim 23, wherein the correction factors are determined by a numerical analysis of the data representative of the light emerging from the interaction spaces generated.

26. The method as claimed in claim 1, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected in real time contemporaneously with the recording of the signals from the interaction space.

27. The method as claimed in claim 1, wherein the interaction space is an interaction layer at a surface of a fixed carrier, the areal extent of said interaction layer being defined by the interaction area with the impinging power-modulated excitation light and its depth being defined by the range of the modulated excitation light intensity in this space dimension perpendicular to said surface of the carrier.

28. The method as claimed in claim 27, wherein compounds or substances or molecular subgroups are situated within the interaction space which, under the action of the excitation light, are capable of emitting optical signals correlated nonlinearly therewith, or with the aid of which, after the interaction thereof with further compounds present in the interaction space, optical signals correlated nonlinearly with the excitation light can be generated.

29. The method as claimed in claim 27, wherein one or a plurality of specific binding partners for the detection of one or a plurality of analytes are immobilized on the surface of said fixed carrier in a binding assay, the analyte detection being effected on the basis of an optical response signal, correlated nonlinearly with the excitation light power, of the immobilized binding partner itself or of a binding partner supplied in solution for binding to the immobilized binding partner or of one or a plurality of further binding partners supplied in one or a plurality of additional method steps.

30. The method as claimed in claim 29, wherein the specific binding partners immobilized on the surface of said fixed carrier are the one or the plurality of analytes themselves which are immobilized, wherein the one or the plurality of analytes are embedded in a native sample matrix or in a sample matrix that is modified by one or a plurality of conditioning steps.

31. The method as claimed in claim 29, wherein the specific binding partners immobilized on the surface of said fixed carrier are biological or biochemical or synthetic identification elements for the specific identification of one or a plurality of analytes situated in a supplied sample.

32. The method as claimed in claim 29, wherein said binding partners are selected from the group consisting of proteins, peptides, enzymes, glycopeptides, oligosaccharides, lectins, antigens for antibodies, proteins functionalized with additional binding sites, nucleic acids, nucleic acid analogs, aptamers, membrane-bound and isolated receptors and ligands thereof, cavities produced by chemical synthesis for receiving molecular imprints, natural polymers and synthetic polymers.

33. The method as claimed in claim 28, wherein the compounds or substances or molecular subgroups are applied on the surface of said fixed carrier, and are immobilized in discrete measurement regions which may have an arbitrary geometry wherein an individual measurement region can optionally contain identical or different compounds or substances or molecular subgroups or specific binding partners.

34. The method as claimed in claim 33, wherein discrete measurement regions are produced by spatially selective application of specific binding partners on said fixed carrier or of compounds or substances or molecular subgroups which, under the action of the excitation light, are capable of emitting optical signals correlated nonlinearly therewith, or with the aid of which, after the interaction thereof with further compounds present in the interaction space, optical signals correlated nonlinearly with the excitation light can be generated.

35. The method as claimed in claim 33, wherein there are applied between the spatially separate measurement regions or in unoccupied partial regions within said measurement regions compounds that are chemically neutral with respect to the analytes and/or with respect to binding partners.

36. The method as claimed in claim 28, wherein, at the surface of said fixed carrier, the compounds or substances or molecular subgroups are applied, or specific binding partners are applied wherein such are immobilized directly or by means of a spacer formed as an independent molecule or molecular group at the surface of said fixed carrier, with utilization of one or a plurality of types of interactions from the group of interactions consisting of hydrophilic interactions, electrostatic interactions and covalent binding.

37. The method as claimed in claim 28, wherein an adhesion promoting layer is applied between the surface of said fixed carrier and the immobilized compounds or substances or molecular subgroups or the applied specific binding partners.

38. The method as claimed in claim 33, wherein more than 10 measurement regions are arranged on a square centimeter in a two-dimensional arrangement on the surface of said fixed carrier.

39. The method as claimed in claim 27, wherein said fixed carrier is optically transparent at the wavelength of an acting excitation light.

40. The method as claimed in one of claim 27, wherein said fixed carrier is essentially planar.

41. The method as claimed in claim 27, wherein said fixed carrier comprises an optical waveguide structure comprising one or a plurality of layers.

42. The method as claimed in claim 27, wherein said fixed carrier comprises a planar optical waveguide that is continuous or divided into discrete wave-guiding regions comprising one or a plurality of layers.

43. The method as claimed in claim 27, wherein said fixed carrier comprises a planar optical thin-film waveguide with an essentially optically transparent, wave-guiding layer (a) on a second, essentially optically transparent layer (b) having a lower refractive index than layer (a) and optionally an essentially optically transparent intermediate layer (b′

) between layer (a) and layer (b) having a lower refractive index than layer (a).

44. The method as claimed in claim 41, wherein a wave-guiding layer of said fixed carrier is in optical contact with one or a plurality of optical coupling elements which enable excitation light to be coupled into said wave-guiding layer, said optical coupling elements being selected from the group consisting of prism couplers, evanescent couplers with united optical waveguides with overlapping evanescent fields, end face couplers with focusing lenses arranged before an end side of said wave-guiding layer of the evanescent field sensor platform, and grating couplers.

47. The method as claimed in claim 1, wherein said data representative of the portion of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light intensity comprise the data representative of signals of a frequency doubling, summation or differential frequency generation.

48. The method as claimed in claim 1, wherein said data representative of the portion of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light intensity are induced by a multi-photon absorption.

49. The method as claimed in claim 48, wherein said multiphoton absorption is a two-photon absorption.

17. The method as claimed in 1, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a parallel series expansion.

22. The method as claimed in 1, wherein in case of a modulation of the excitation light power experimentally dictated deviations of the excitation light powers from the desired values provided for the modulation are compensated for using numerical corrections.

45. The method as claimed in 41, wherein a wave-guiding layer of the fixed carrier comprises one or a plurality of grating structures (c) which enable excitation light to be coupled in.

46. The method as claimed in claim 45, wherein a wave-guiding layer of the fixed carrier comprises one or a plurality of grating structures (c′

) having an identical or different grating period and grating depth with respect to grating structures (c) and enable light guided in said wave-guiding layer to be coupled out.

50. An analytical system for highly sensitive simultaneous measurement of nonlinear optical emission signals, spatially resolved in one or two spatial dimensions, comprising:

at least one light source for emitting excitation light,

technical auxiliary means for power modulation and/or pulse duration modulation of the excitation light emerging from the at least one light source,

an interaction volume or an interaction area or an interaction layer, designated jointly as interaction space, wherein one or a plurality of emissions that are correlated nonlinearly with the excitation light can be excited,

at least one one- or two-dimensional detector array for measuring the light emerging from the interaction space,

a computer to which the measurement data of said detector arrays are transmitted and with the aid of which the measurement data are formatted in a one- or multidimensional data matrix and analyzed,

wherein data representative of those portions of the light emerging from the interaction spaces which are linearly proportional to the intensity of the excitation light available in the interaction spaces are separated from data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the available excitation light intensity.

51. The analytical system as claimed in claim 50, wherein the method does not comprise any components for a spectral filtering of the light that is to be detected and emerge from the interaction spaces.

52. The analytical system as claimed in claim 50, wherein the method additionally comprises components for a spectral filtering of the light that is to be detected and emerge from the interaction spaces.

53. The analytical system as claimed in claim 50, wherein at least one one- or two-dimensional detector array is selected from the group consisting of CCD cameras, CCD chips, CMOS cameras, CMOS chips, photodiode arrays, avalanche diode arrays, multichannel plates and multichannel photomultipliers, such that a phase-sensitive demodulation is capable of being integrated into said detector array.

54. The analytical system as claimed in claim 50, wherein said technical auxiliary means for the modulation of the excitation light radiated in to an interaction space are selected from the group consisting of optomechanical, acousto-optical and electro-optically active auxiliary means.

55. The analytical system as claimed in claim 54, wherein said optomechanical and/or acousto-optical and/or electro-optically active auxiliary means are selected from the group consisting of mechanical shutters and rotating choppers which in each case alternately block and release the light path between the excitation light source and the interaction space, polarization-selective components, acousto-optical modulators and modulators based on interference effects.

56. The analytical system as claimed in claim 50, wherein the modulation of the excitation light radiated into an interaction space is effected by means of direct, active modulation of the light radiated from the excitation light source.

57. The analytical system as claimed in claim 56, wherein the modulation of the excitation light radiated in to an interaction space is effected by means of modulation of the excitation current for a semiconductor laser as excitation light source.

58. The analytical system as claimed in claim 50, wherein the modulation of the excitation light radiated into an interaction space is effected periodically.

59. The analytical system as claimed in claim 50, wherein the modulation of the excitation light radiated into an interaction space is effected non-periodically.

60. The analytical system as claimed in claim 50, wherein the modulation of the excitation light radiated into an interaction space consists of modulation of the intensity radiated in.

61. The analytical system as claimed in claim 50, wherein the modulation of the excitation light radiated into an interaction space consists of simultaneous modulation of pulse duration and peak power of the excitation light radiated in.

62. The analytical system as claimed in claim 50, wherein the system is effected without detection of the modulated excitation light or a measurement variable proportional thereto.

63. The analytical system as claimed claim 50, wherein the system comprises, in addition to the detection of the light emerging from the interaction spaces, a detection of the modulated excitation light or a measurement variable proportional thereto.

64. The analytical system as claimed in claim 50, wherein the detection of the light emerging from the interaction spaces is effected in a manner temporally correlated with the modulation of the excitation light power.

65. The analytical system as claimed in claim 64, wherein the detection of the light emerging from the interaction spaces is effected with a frequency corresponding to an integer multiple of the modulation frequency of the excitation light power.

66. The analytical system as claimed in claim 50, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a parallel series expansion.

67. The analytical system as claimed in claim 50, wherein the separation of the data representative of the portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a harmonic analysis.

68. The analytical system as claimed in claim 50, wherein the separation of the data representative of the portions of light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a stepped modulation of the excitation light power.

69. The analytical system as claimed in claim 50, wherein the separation of the data representative of the portions of light emerging from the interaction spaces which are nonlinearly proportional to the excitation light power from the data representative of the remaining portions of said light is effected using a four-step algorithm for the modulation of the excitation light power.

70. The analytical system as claimed in claim 50, wherein the interaction space is an interaction layer at a surface of a fixed carrier, the areal extent of the interaction layer being defined by the interaction area with the impinging power-modulated excitation light and its depth being defined by the range of the modulated excitation light intensity in this space dimension perpendicular to said surface of the carrier.

71. The analytical system as claimed in claim 70, wherein there are situated within the interaction space compounds or substances or molecular subgroups which, under the action of the excitation light, are capable of emitting optical signals correlated nonlinearly therewith, or with the aid of which, after the interaction thereof with further compounds present in the interaction space, optical signals correlated nonlinearly with the excitation light can be generated.

72. The analytical system as claimed in claim 70, wherein there are immobilized on the surface of said fixed carrier one or a plurality of specific binding partners for the detection of one or a plurality of analytes in a binding assay the analyte detection being effected using an optical response signal, correlated nonlinearly with the excitation light power, of the immobilized binding partner itself or of the binding partner supplied in solution for binding to the immobilized binding partner or of one or a plurality of further binding partners supplied in one or a plurality of additional method steps.

73. The analytical system as claimed in claim 72, wherein the specific binding partners immobilized on the surface of said fixed carrier are the one or the plurality of analytes themselves which are immobilized, wherein the specific binding partners are embedded in a native sample matrix or in a sample matrix that is modified by one or a plurality of conditioning steps.

74. The analytical system as claimed in claim 72, wherein the specific binding partners immobilized on the surface of said fixed carrier are biological or biochemical or synthetic identification elements for the specific identification of one or a plurality of analytes situated in a supplied sample.

75. The analytical system as claimed in claim 72, wherein said binding partners are selected from the group consisting of proteins, peptides, enzymes, glycopeptides, oligosaccharides, lectins, antigens for antibodies, proteins functionalized with additional binding sites, nucleic acids, nucleic acid analogs, aptamers, membrane-bound and isolated receptors and ligands thereof, cavities produced by chemical synthesis for receiving molecular imprints, natural polymers and synthetic polymers.

76. The analytical system as claimed in claim 71, wherein the compounds or substances or molecular subgroups are applied on the surface of said fixed carrier, and are immobilized in discrete measurement regions which may have an arbitrary geometry, wherein an individual measurement region can optionally contain identical or different compounds or substances or molecular subgroups or specific binding partners.

77. The analytical system as claimed in claim 76, wherein more than 10 measurement regions are arranged on a square centimeter in a two-dimensional arrangement on the surface of said fixed carrier.

78. The analytical system as claimed in claim 70, wherein said fixed carrier is optically transparent at a wavelength of an acting excitation light.

79. The analytical system as claimed in claim 70, wherein said fixed carrier is essentially planar.

80. The analytical system as claimed in claim 70, wherein said fixed carrier comprises an optical waveguide structure, comprising one or a plurality of layers.

81. The analytical system as claimed in claim 70, wherein said fixed carrier comprises a planar optical waveguide that is continuous or divided into discrete wave-guiding regions and comprises one or a plurality of layers.

82. The analytical system as claimed in claim 70, wherein said fixed carrier comprises a planar optical thin-film waveguide with an essentially optically transparent, wave-guiding layer (a) on a second, essentially optically transparent layer (b) having a lower refractive index than layer (a) and optionally an essentially optically transparent intermediate layer (b′

) between layer (a) and layer (b) having a lower refractive index than layer (a).

83. The analytical system as claimed in claim 80, wherein a wave-guiding layer of said fixed carrier is in optical contact with one or a plurality of optical coupling elements which enable excitation light to be coupled into said wave-guiding layer, said optical coupling elements being selected from the group of consisting of prism couplers, evanescent couplers with united optical waveguides with overlapping evanescent fields, end face couplers with focusing lenses arranged before an end side of said wave-guiding layer of the evanescent field sensor platform, and grating couplers.

84. The analytical system as claimed in claim 80, wherein a wave-guiding layer of the fixed carrier comprises one or a plurality of grating structures (c) which enable excitation light to be coupled in.

85. The analytical system as claimed in claim 80, wherein a wave-guiding layer of the fixed carrier comprises one or a plurality of grating structures (c′

) having an identical or different grating period and grating depth with respect to grating structures (c) and enable light guided in said wave-guiding layer to be coupled out.

86. The analytical system as claimed in claim 50, wherein said portions of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light intensity comprise the signals of a frequency doubling, summation or differential frequency generation.

87. The analytical system as claimed in claim 50, wherein said data representative of the portion of the light emerging from the interaction spaces which are nonlinearly proportional to the excitation light intensity are induced by a multi-photon absorption.

88. The analytical system as claimed in claim 87, wherein said multiphoton absorption is a two-photon absorption.