Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources

• US 20080030628A1
• Filed: 02/09/2007
• Published: 02/07/2008
• Est. Priority Date: 02/13/2006
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Zaccarin, Denis, Turner, Stephen, Foquet, Mathieu, Lundquist, Paul, Maxham, Mark, Lacroix, Yves
• Granted Patent: US 7,995,202 B2
• Time in Patent Office: Days
• US Class Current: 348/751
• CPC Class Codes: G01J 3/02 Details
• CPC Class Codes: G01J 3/0224 using polarising or depolarising elements
• CPC Class Codes: G01J 3/10 Arrangements of light sources specially adapted for spectrometry or colorimetry
• CPC Class Codes: G01J 3/14 using refracting elements, e.g. prisms G01J3/18, G01J3/26 take precedence prisms per se G02B5/04
• CPC Class Codes: G01J 3/2803 using photoelectric array detector
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 21/6445 Measuring fluorescence polarisation
• 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
• CPC Class Codes: G01N 2201/126 Microprocessor processing

Abstract

Methods and systems for real-time monitoring of optical signals from arrays of signal sources, and particularly optical signal sources that have spectrally different signal components. Systems include signal source arrays in optical communication with optical trains that direct excitation radiation to and emitted signals from such arrays and image the signals onto detector arrays, from which such signals may be subjected to additional processing.

Claims

1. A method of monitoring one or more optical signal from a substrate having at least a first signal source disposed thereon, comprising:

imaging the optical signal onto an imaging detector that comprises a plurality of pixels;

subjecting signal data from a first set of pixels to a first data process, wherein the first set of pixels correspond to at least a portion of the imaged signal;

subjecting signal data from a second set of pixels different from the first set of pixels to a second data process different from the first data process; and

recording an output from the first data process to monitor the optical signal.

2. The method of claim 1, wherein the first set of pixels corresponds to a first portion of pixels upon which the optical signal is imaged.

3. The method of claim 2, wherein the first portion of pixels correspond to a portion of the optical signal having an intensity above a threshold intensity.

4. The method of claim 2, wherein the first portion of pixels correspond to a central portion of the optical signal.

5. The method of claim 2, wherein the second set of pixels comprises a portion of pixels upon which the optical signal is imaged, but which are not within the first portion of pixels.

6. The method of claim 2, wherein the second set of pixels comprises pixels upon which the optical signal is not imaged.

7. The method of claim 1, wherein the second set of pixels comprise pixels between an image of a first optical signal imaged onto the detector and a second optical signal imaged onto the detector.

8. The method of claim 1, wherein the first set of pixels comprises pixels in a first row or column of pixels and the second set of pixels comprises pixels in a second row of or column of pixels different from the first row of pixels.

9. The method of claim 1, wherein the first set of pixels comprises a first plurality of rows or columns of pixels, and the second set of pixels comprises a second plurality of rows or columns of pixels, wherein the rows or columns of pixels in the second plurality of rows or columns of pixels are positioned between the rows or columns of pixels in the first plurality of rows or columns of pixels.

10. The method of claim 1, wherein one or more rows or columns of pixels in the first plurality of rows or columns of pixels or the second plurality of rows or columns of pixels is at least two pixels in width.

11. The method of claim 1, wherein each row or column of pixels is at least two pixels in width.

12. The method of claim 1, wherein the first data process comprises extracting signal data from the detector and processing the data through a gain register to amplify the data associated with the optical signal.

13. The method of claim 1, wherein the second data process discards signal data from the second set of pixels without extracting the signal data from the detector.

14. The method of claim 1, wherein the second data process comprises discarding the signal data from the second set of pixels prior to processing the signal data through a gain register.

15. The method of claim 1, wherein the at least first signal source comprises a reaction region on the substrate having at least a first fluorescently labeled reactant that emits the optical signal in response to an excitation radiation.

16. A system for monitoring optical signals, comprising:

a substrate having at least a first source of optical signals disposed thereon;

an optical train positioned to receive optical signals from the at least first source of optical signals and image the optical signals onto a imaging detector;

an imaging detector comprising a plurality of pixels, the detector positioned to receive the image of the optical signals on a first set of pixels in the plurality of pixels; and

a processor programmed to process signal data from the detector to monitor the optical signals;

wherein at least one of the detector or processor are configured to process signal data from the first set of pixels in a first data process and data from a second set of pixels in the plurality of pixels different from the first set of pixels in a second data process different from the first data process.

17. The system of claim 16, wherein the first data process comprises subjecting the signal data from the first set of pixels to a gain protocol to amplify the signal data and wherein the second data process does not include subjecting signal data from the second set of pixels to the gain protocol to amplify the signal data.

18. The system of claim 17, wherein the detector comprises a gain register configured to run the gain protocol.

19. The system of claim 16, wherein the second data process does not comprise extracting signal data from the second set of pixels.

20. The system of claim 16, wherein the second data process comprises combining signal data from the second set of pixels with signal data from a plurality of other pixels in the detector upon which signal is not incident to provide combined signal data, and concurrently processing the combined signal data.

21. The system of claim 16, wherein the second data process comprises discarding the combined signal data.

22. The system of claim 20, wherein the second data process comprises subjecting the combined signal data to a gain protocol to amplify the combined signal data.

23. The system of claim 16, wherein the substrate comprises a plurality of discrete signal sources disposed thereon.

24. The system of claim 23, wherein each of the plurality of signal sources comprises a fluorescently labeled compound that emits the optical signals in response to an excitation radiation, and wherein the system further comprises a source of excitation radiation positioned to direct excitation radiation at the plurality of signal sources on the substrate.

25. The system of claim 23, wherein the substrate comprises a plurality of reaction wells, each reaction well comprising a complex of a nucleic acid polymerase, a template nucleic acid sequence, a primer sequence and at least one fluorescently labeled nucleotide or nucleotide analog the emits the optical signal in response to activation radiation directed at the reaction well.

26. The system of claim 25, wherein the reaction well comprises a confined illumination region.

27. The system of claim 26, wherein the reaction well comprises a zero mode waveguide.

28. A method of monitoring an optical signal from a source of optical signals, comprising:

imaging the optical signal onto a plurality of pixels on an imaging detector;

combining signal data from the plurality of pixels; and

processing the combined signal data to monitor the optical signal.

29. The method of claim 28, wherein the plurality of pixels comprises at least 4 pixels.

30. The method of claim 28, wherein the plurality of pixels comprises at least 10 pixels.

31. The method of claim 28, wherein the plurality of pixels comprises at least 20 pixels.

32. The method of claim 28, wherein the source of optical signals comprises a reaction region on a substrate, the reaction region comprising one or more fluorescently labeled reactants that emit the optical signals in response to an excitation radiation.

33. The method of claim 28, wherein the source of optical signals comprises at least two spectrally resolvable optical signals, and further comprising separately imaging each of the spectrally resolvable signals onto a spatially distinct plurality of pixels.

34. The method of claim 28, wherein the source of optical signals comprises a reaction region on a substrate, the reaction region comprising at least two different fluorescently labeled reactants that emit at least two spectrally resolvable optical signals in response to an excitation radiation.

35. The method of claim 34, wherein the reaction region comprises a confined region on a substrate.

36. The method of claim 35, wherein the confined region comprises a reaction well.

37. The method of claim 36, wherein the confined region comprises a zero mode waveguide.

38. The method of claim 34, wherein the at least two different fluorescently labeled reactants comprise fluorescently labeled nucleotides or nucleotide analogs.

39. The method of claim 28, wherein the source of optical signals comprises:

a reaction region having a complex of a nucleic acid polymerase, a template nucleic acid and a primer sequence immobilized therein, and a plurality of different nucleotide analogs, where each different nucleotide analog includes a different fluorescently label that is optically resolvable by its emission spectrum; and

wherein the optical signals comprise fluorescent emissions from the different nucleotide analogs upon incorporation in polymerase mediated, template dependent extension of the primer.

40. A system for monitoring optical signals, comprising:

a substrate having at least a first source of optical signals disposed thereon;

an optical train positioned to receive optical signals from the at least first source of optical signals and image the optical signals onto an imaging detector;

an imaging detector comprising a first plurality of pixels, the detector positioned to receive the image of the optical signals on a second plurality of a pixels in the first plurality of pixels; and

a processor programmed to process signal data from the detector to monitor the optical signals;

wherein at least one of the imaging detector and processor are configured to combine signal data from the second plurality of pixels to provide combined signal data, and process the combined signal data.

41. The system of claim 40, wherein the detector is configured to combine the signal data from the second plurality of pixels, prior to subjecting the data to a gain protocol.

42. The system of claim 40, wherein the detector comprises a gain register, and the detector is configured to provide the combined signal data and pass the combined signal data through the gain register.

43. A method of monitoring a plurality of spectrally distinct optical signals from a source of optical signals, comprising:

passing the plurality of optical signals through an optical train that is configured to image each of the plurality of spectrally distinct optical signals onto an imaging detector, wherein an image of each spectrally distinct optical signal has an image shape characteristic of its spectral characteristics;

imaging the plurality of optical signals onto the imaging detector; and

identifying each optical signal by its characteristic image shape to monitor the plurality of spectrally distinct optical signals.

44. The method of claim 43, wherein the plurality of spectrally distinct optical signals comprises at least two different fluorescently labeled compounds in the source of optical signals, each different fluorescently labeled compound having a different emission spectrum when exposed to excitation radiation.

45. The method of claim 43, wherein the plurality of spectrally distinct optical signals comprises at least four different fluorescently labeled compounds in the source of optical signals, each different fluorescently labeled compound having a different emission spectrum when exposed to excitation radiation.

46. The method of claim 45, wherein the at least four different fluorescently labeled compounds comprise at least four different fluorescently labeled nucleotides, each different nucleotide being labeled with a different fluorescent label having a different emission spectrum.

47. The method of claim 43, wherein the imaging detector is selected from a CCD, an EMCCD, and ICCD, a CMOS sensor, and a hybrid CMOS/CCD sensor.

48. A system for monitoring a plurality of different optical signals from a source of optical signals, comprising:

a substrate having at least a first source of optical signals disposed thereon, the source of optical signals including a plurality of spectrally distinct optical signals;

an optical train positioned to receive the plurality of different optical signals from the at least first source of optical signals and differentially image each of the plurality of different optical signals onto an imaging detector such that an image of each of the different optical signals is characteristic of the spectrally distinct optical signal;

an imaging detector; and

a processor for processing signal data from the imaging detector, wherein the processor is configured to identify the spectrally distinct optical signal by its characteristic image shape on the imaging detector.

49. The system of claim 48, wherein the at least first source of optical signals comprises a reaction region having a plurality of different fluorescently labeled compounds disposed therein, each of the different fluorescently labeled compounds having a distinct fluorescent emission spectrum in response to excitation radiation.

50. A method of processing an optical image on an imaging detector from a source of optical signals, comprising:

imaging the optical signal onto an array of pixels on an imaging detector;

acquiring signal data from the plurality of pixels upon which the optical signal is imaged;

transferring the acquired signal data to a storage region of the detector; and

subjecting the acquired data to a gain process during the transferring step to amplify the signal data.

51. The method of claim 50, wherein the signal data is amplified by at least 100 fold.

52. The method of claim 50, wherein the signal data is amplified by at least 500 fold.

53. The method of claim 50, wherein the signal data is amplified by at least 1000 fold.

54. A system for monitoring optical signals, comprising:

a substrate having at least a first source of optical signals disposed thereon;

an optical train positioned to receive optical signals from the at least first source of optical signals and image the optical signals onto an imaging detector; and

an imaging detector comprising;

a plurality of optically active pixels in an image acquisition portion of the detector;

a data storage portion of the detector operably coupled to the image acquisition portion to receive signal data from the image acquisition portion in a frame transfer process; and

wherein the detector is configured to apply a gain voltage to the signal data during the frame transfer process to amplify the signal data transferred to the data storage portion of the detector.

55. An imaging detector, comprising:

a plurality of optically active pixels in an image acquisition portion of the detector;

a data storage portion of the detector operably coupled to the image acquisition portion to receive signal data from the image acquisition portion in a frame transfer process; and

wherein the detector is configured to apply a gain voltage to the signal data during the frame transfer process to amplify the signal data transferred to the data storage portion of the detector.

56. The imaging detector of claim 55, wherein the image acquisition portion of the detector is selected from a CCD, a CMOS sensor array and a hybrid CCD/CMOS sensor array.

57. A method of monitoring optical signals, comprising:

imaging the optical signals onto an imaging detector that comprises a plurality of pixels;

selecting signal data from the plurality of pixels that falls within a selected signal amplitude range; and

subjecting the signal data selected in the selecting step to a gain protocol to amplify the selected signal data, while not amplifying signal data that was not selected.

58. An imaging detector, comprising:

a plurality of optically active pixels in an image acquisition portion of the detector;

a data storage portion of the detector operably coupled to the image acquisition portion to receive signal data from the image acquisition portion in a frame transfer process;

a gain register operably coupled to the data storage portion to amplify signal data from the data storage portion;

wherein the detector is configured to pass signal data through the gain register that falls within a selected signal amplitude range.

59. A method of monitoring optical signals in a system that comprises:

a source of optical signals;

an optical train positioned to receive the optical signals from the source of optical signals and image the optical signals onto an imaging detector; and

an imaging detector positioned to receive imaged optical signals onto a plurality of optically sensitive pixels that are operably coupled to a gain register to amplify signal data from the plurality of pixels;

the method comprising;

measuring a gain from the gain register in the absence of an imaged optical signal on the plurality of optical signals.

60. The method of claim 59, further comprising calibrating the gain register to provide a gain in a desired gain range, based upon the gain measured in the measuring step.

61. A method of monitoring a plurality of spectrally different optical signals from a single signal source, comprising:

collecting the spectrally different optical signals in an optical train;

transmitting the spectrally different optical signals through the optical train that is configured to differentially image each of the spectrally different optical signals onto an imaging detector;

differentially imaging each of the spectrally different optical signals onto the imaging detector; and

identifying each spectrally different signal imaged upon the detector by its image on the imaging detector.

62. A method of monitoring optical signals from a plurality of signal sources on a substrate, comprising:

imaging the plurality of signal sources onto an imaging detector that comprises a plurality of pixels, wherein images of the plurality of signal sources are directed substantially onto a first subset of the plurality of pixels, but not substantially on a second subset of the plurality of pixels; and

processing signal data from the first subset of pixels but not from the second subset of pixels, to monitor signals from the plurality of signal sources.

63. The method of claim 62, wherein the first subset of the plurality of pixels comprises a first row or column of pixels on the imaging detector and the second subset of the plurality of pixels comprises a second row or column of pixels on the imaging detector, respectively.

64. A method of monitoring one or more signals from a signal source, comprising:

imaging a first optical signal onto a first plurality of pixels on an imaging detector;

selecting from the first plurality of pixels a first subset pixels that meet or exceed a signal quality threshold; and

processing data from the first subset of pixels to monitor the one or more signals from the signal source.

65. The method of claim 64, wherein the signal source comprises a substrate having a plurality of discrete signal sources disposed thereon.

66. The method of claim 64, wherein the signal source comprises a plurality of different fluorescently labeled compounds, each of the plurality of different fluorescently labeled compounds emitting a fluorescent signal in response to excitation radiation that is different from each other of the plurality of different fluorescently labeled compounds.

67. A method of monitoring one or more optical signals from one or more discrete signal sources, comprising:

imaging the one or more signals onto a plurality of pixels on a detector array;

selecting a subset of the plurality of pixels;

recording data from the subset of the plurality of pixels as indicative of the one or more signals;

correlating the data to a signal from the discrete signal source.

68. A method of monitoring a plurality of optical signals from one or more discrete signal sources, comprising:

imaging the plurality of signals onto a detector array, wherein each signal is imaged onto a plurality of pixels on the detector array; and

processing data from the plurality of pixels while discarding data from pixels not in the plurality of pixels.

69. A system, comprising:

an array of optical signal sources, each signal source being capable of emitting a plurality of signals having different optical wavelengths;

an optical train for collecting the signals from the array of signal sources and differentially imaging each of the plurality of signals having different optical wavelengths onto a detector; and

a detector for detecting the signals imaged thereon.

70. A system, comprising:

an array of a plurality of optical signal sources, the plurality of optical signal sources having a plurality of spectrally resolvable fluorescent compounds associated therewith;

a source of excitation radiation;

a detector array; and

an optical train configured to;

direct excitation radiation from the source of excitation radiation to the array of signal sources;

receive emitted fluorescent signals from the array of signal sources; and

image the fluorescent signals onto the detector array, wherein the optical train is characterized by a dichroic filter in optical communication with an objective lens, wherein the dichroic filter is reflective of the fluorescent signals and transmissive to the excitation radiation.

71. A method for resolvably detecting a plurality of spectrally different optical signals from at least a first signal source, comprising:

collecting the plurality of spectrally different optical signals from the signal source; and

differentially imaging each of the spectrally different optical signals on a detector array, such that each different signal is resolvably detected.

72. A method of monitoring an optical signal from a signal source, comprising:

imaging the optical signal onto a plurality of pixels of a detector array in a signal image;

selecting a subset of the plurality of pixels in the signal image having a higher signal intensity within the signal image than other pixels within the signal image;

measuring the signal in the subset of pixels.

73. A method of monitoring signals from a plurality of signal sources, comprising:

imaging each of the plurality of signals onto a detector array comprising a plurality of rows and columns of pixels;

processing data derived from pixels in rows or columns upon which the plurality of signals is imaged, but not from rows of pixels upon which no signal is imaged.

74. The method of claim 73, further comprising processing data only from pixels upon which the plurality of signals are imaged.

75. The method of claim 73, wherein the processing step comprises gain processing.

76. The method of claim 73, wherein the processing step comprises subtracting background signal data.

77. The method of claim 73, wherein the [processing step comprises recording the data.

78. The method of claim 73, wherein the processing step comprises correlating data from a signal with an event within the signal source.

79. The method of claim 73, wherein the event within the signal source comprises a given chemical reaction.

80. The method of claim 73, wherein the given chemical reaction comprises a template directed incorporation of a nucleotide or nucleotide analog into a nascent nucleic acid strand by a polymerase enzyme.

81. A method of processing signals imaged onto an EMCCD, comprising:

determining whether the signals imaged onto the EMCCD are within a preselected signal amplitude range; and

processing only signals that are within the preselected amplitude range through a gain register on the EMCCD.

82. A method of processing signals imaged onto a CCD that is configured to transfer image data acquired by the detector to a storage region on the CCD in a frame transfer process, comprising applying an elevated voltage to the frame transfer process to amplify signal data being transferred.

83. A system for monitoring optical signals from a plurality of sources of optical signals, comprising:

an array of discrete sources of optical signals, said discrete sources emitting optical signals having different spectral characteristics;

an excitation radiation source;

a detector array;

an optical train configured to;

direct excitation radiation from the source of excitation radiation to the array of discrete sources;

receive emitted optical signals from the array of signal sources; and

differentially image the optical signals having different spectral characteristics onto a detector array; and

a processor configured to record the optical signals imaged onto the detector array and correlate the optical signals with a property of or an event occurring within the sources of optical signals.