Method and system for microfluidic device and imaging thereof

• US 7,695,683 B2
• Filed: 05/20/2004
• Issued: 04/13/2010
• Est. Priority Date: 05/20/2003
• Status: Expired due to Fees

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Lee, Michael, Quan, Emerson Chueng, Sun, Gang, Cesar, Christopher G., Harris, Greg, Taylor, Colin Jon
• Primary Examiner(s): Siefke; Sam P
• Application Number:
  US10/851,777
• Publication Number:
  US 20090299545A1
• Time in Patent Office: 2,154 Days
• Field of Search: 422/50, 422/68.1, 422/100, 436/43, 436/63, 382/100, 382/141, 382/152, 382/154, 382/312
• US Class Current: 422/504
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: G01N 33/00 Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
• CPC Class Codes: Y10T 436/11 Automated chemical analysis

Abstract

A biological substrate, e.g., microfluidic chip. The substrate includes a rigid substrate material, which has a surface region capable of acting as a handle substrate. The substrate also has a deformable fluid layer coupled to the surface region. One or more well regions are formed in a first portion of the deformable fluid layer and are capable of holding a fluid therein. The one or more channel regions are formed in a second portion of the deformable fluid layer and are coupled to one or more of the well regions. An active region is formed in the deformable fluid layer. At least three fiducial markings are formed within the non-active region and disposed in a spatial manner associated with at least one of the well regions. A control layer is coupled to the fluid layer.

Claims

1. A method for processing a microfluidic device, the method comprising:

providing a flexible substrate including a first plurality of fiducial markings, wherein in a first state, the first plurality of fiducial markings correspond to a first plurality of design locations respectively, the first plurality of design locations having a first spatial relationship with respect to each other;

determining a first plurality of actual locations corresponding to the first plurality of fiducial markings respectively, wherein in a second state, one or more of the first plurality of actual locations are shifted from the first plurality of design locations such that the first plurality of actual locations have a second spatial relationship with respect to each other different from the first spatial relationship;

processing information associated with the first plurality of actual locations and the first plurality of design locations;

determining a transformation between a design space and a measurement space based on at least information associated with the first plurality of actual locations and the first plurality of design locations, the design space associated with the first plurality of design locations, the measurement space associated with the first plurality of actual locations;

performing a first alignment to the flexible substrate based on at least information associated with the transformation between the design space and the measurement space;

acquiring a first plurality of images of a first fiducial marking;

processing information associated with the first plurality of images;

performing a second alignment to the flexible substrate based on at least information associated with the first plurality of images; and

acquiring a second image of the flexible substrate.

2. The method of claim 1 wherein the flexible substrate further comprises a first chamber, the first chamber being capable of holding a fluid therein.

3. The method of claim 2 wherein the first fiducial marking is within a vicinity of the first chamber.

4. The method of claim 3 wherein the second image of the flexible substrate is associated with the first chamber.

5. The method of claim 4, and further comprising providing the flexible substrate on a stage.

6. The method of claim 4, and further comprising storing the second image in a memory.

7. The method of claim 6 wherein the second image comprises 3900 by 3030 pixels.

8. The method of claim 7 wherein the second image comprises a 16 bit image.

9. The method of claim 8 wherein the memory is a computer memory.

10. The method of claim 1 wherein the first plurality of fiducial markings comprises three fiducial markings.

11. The method of claim 10 wherein the first plurality of fiducial markings is free from the first fiducial marking.

12. The method of claim 11 wherein the processing information associated with the first plurality of images comprises:

determining a first plurality of focus scores associated with the first plurality of images;

processing information associated with the first plurality of focus scores;

determining a focus position based on at least information associated with the first plurality of focus scores.

13. The method of claim 12 wherein the determining a first plurality of focus scores associated with the first plurality of images comprises:

for each of the first plurality of images,

determining a first value associated with a first characteristic of the each of the first plurality of images;

blurring the each of the first plurality of images;

determining a second value associated with the first characteristic of the blurred each of the first plurality of images;

if the second value is equal to or larger than a predetermined value, determining a focus score equal to a ratio between the first value and the second value;

if the second value is smaller than the predetermined value, determining the focus score equal to a ratio between the first value and the predetermined value.

14. The method of claim 13 wherein the acquiring a first plurality of images of the first fiducial marking comprises:

moving the flexible substrate to a first plurality of positions;

for each of the first plurality of positions, acquiring one of the first plurality of images.

15. The method of claim 11 wherein the performing a second alignment to the flexible substrate comprises moving the flexible substrate to the focus position.

16. The method of claim 1, and further comprising:

acquiring a first image of the first fiducial marking associated with the flexible substrate;

performing a third alignment to the flexible substrate based on at least information associated with the first image.

17. The method of claim 16 wherein the acquiring a first image of the first fiducial marking comprises:

acquiring a third image;

processing information associated the third image;

determining whether the first fiducial marking is present in the third image;

if the first fiducial marking is not present in the third image, translate the flexible substrate in at least one dimension;

wherein the third image is the first image if the first fiducial marking is present in the third image;

wherein the processing information associated the third image includes;

segmenting the third image;

performing blob analysis to the third image.

83. The method of claim 1 wherein the first plurality of actual locations are determined sequentially.

18. A method for processing a microfluidic device, the method comprising:

providing a flexible substrate including at least three fiducial markings, a first additional fiducial marking, and a first chamber capable of holding a fluid therein, wherein in a first state, the at least three fiducial markings correspond to at least three design locations respectively, the at least three design locations having a first spatial relationship with respect to each other;

obtaining information associated with at least three actual locations of the at least three fiducial markings respectively, the at least three actual locations associated with a measurement space, wherein in a second state, one or more of the at least three actual locations are shifted from the at least three design locations such that the actual locations have a second spatial relationship with respect to each other different from the first spatial relationship;

determining a transformation between a design space and the measurement space based on at least information associated with the at least three actual locations and the at least three design locations;

performing a first alignment of the flexible substrate based on at least information associated with the transformation between the design space and the measurement space;

acquiring at least a first image of the first additional fiducial marking associated with the first chamber;

performing a second alignment of the flexible substrate based on at least information associated with the first image; and

acquiring a second image of the first chamber associated with the flexible substrate.

19. The method of claim 18 wherein the obtaining information associated with the at least three fiducial markings comprises:

determining the at least three actual locations corresponding to the at least three fiducial markings respectively;

processing information associated with the at least three actual locations and the at least three design locations.

20. The method of claim 18 wherein the acquiring at least a first image of the first additional fiducial marking comprises:

acquiring a first plurality of images of the first additional fiducial marking, the first plurality of images including the first image;

processing information associated with the first plurality of images.

21. The method of claim 18, and further comprising locating the flexible substrate on a stage.

22. The method of claim 21, and further comprising storing the second image in a memory.

23. The method of claim 22 wherein the memory is a computer memory.

24. The method of claim 18 wherein the second image comprises 3900 by 3030 pixels.

25. The method of claim 24 wherein the second image comprises a 16 bit image.

26. The method of claim 18 wherein the performing a second alignment to the flexible substrate comprises:

translating the flexible substrate in at least one dimension to position a chamber in preparation for capturing the second image.

27. The method of claim 18 wherein a volume of the fluid is less than a nanoliter.

28. The method of claim 18 wherein the first additional fiducial marking is a company logo.

29. The method of claim 18 wherein the at least three fiducial markings comprise a company logo.

30. The method of claim 18 wherein the flexible substrate is deformable in three dimensions.

31. The method of claim 30 wherein the flexible substrate is deformed by actions selected from the group consisting of fabrication, handling, and protocols.

32. The method of claim 31 wherein the protocols can result in the flexible substrate swelling or contracting.

33. The method of claim 30 wherein a relationship between the design space and the measurement space is non-planar.

34. The method of claim 33 wherein the flexible substrate is deformed such that a planar transformation is capable to approximately determine an actual location of the first chamber.

35. The method of claim 18, and further comprising:

normalizing the second image;

white balancing the second image;

converting the second image from a first image depth to a second image depth.

36. The method of claim 35 wherein the first image depth is 16 bits and the second image depth is 8 bits.

37. The method of claim 18 wherein the transformation between the design space and the measurement space is non-planar.

38. The method of claim 37 wherein the non-planar transformation comprises a three dimensional parabolic mapping.

39. The method of claim 37 wherein the non-planar transformation is updated using information obtained by characterization of a second additional fiducial marking.

40. A system for processing one or more microfluidic devices, the system including one or more computer-readable media, a stage for locating a flexible substrate, and an imaging system operable to acquire an image of the flexible substrate, the flexible substrate comprising at least three fiducial markings, a first additional fiducial marking, and a first chamber capable of holding a fluid therein, the one or more computer-readable media including:

one or more instructions for providing the flexible substrate;

one or more instructions for obtaining information associated with at least three design locations, wherein in a first state the at least three fiducial markings correspond to the at least three design locations respectively, the at least three design locations having a first spatial relationship with respect to each other and being associated with a design space;

one or more instructions for obtaining information associated with at least three actual locations of the at least three fiducial markings respectively, wherein in a second state one or more of the at least three actual locations are shifted from the at least three design locations such that the at least three actual locations have a second spatial relationship with respect to each other different from the first spatial relationship and are associated with a measurement space;

one or more instructions for determining a transformation between the design space and the measurement space based on at least information associated with the at least three actual locations and the at least three design locations;

one or more instructions for positioning at least one of the stage or the imaging system to perform a first alignment of the flexible substrate based on at least information associated with the transformation between the design space and the measurement space;

one or more instructions for acquiring at least a first image of the first additional fiducial marking associated with the first chamber using the imaging system;

one or more instructions for positioning at least one of the stage or the imaging system to perform a second alignment of the flexible substrate based on at least information associated with the first image; and

one or more instructions for acquiring a second image of the first chamber associated with the flexible substrate using the imaging system.

41. The one or more computer-readable media of claim 40 wherein the one or more instructions for determining a transformation between a design space and a measurement space comprise:

one or more instructions for determining the at least three actual locations corresponding to the at least three fiducial markings respectively;

one or more instructions for processing information associated with the at least three actual locations and the at least three design locations.

42. The one or more computer-readable media of claim 40 wherein the one or more instructions for acquiring at least a first image of the first additional fiducial marking comprise:

one or more instructions for acquiring a first plurality of images of the first additional fiducial marking, the first plurality of images including the first image;

one or more instructions for processing information associated with the first plurality of images.

43. The one or more computer-readable media of claim 40, and further comprising one or more instructions for storing the second image in a memory.

44. The one or more computer-readable media of claim 43 wherein the memory is a computer memory.

45. The one or more computer-readable media of claim 40 wherein the second image comprises 3900 by 3030 pixels.

46. The one or more computer-readable media of claim 45 wherein the second image comprises a 16 bit image.

47. The one or more computer-readable media of claim 40 wherein the one or more instructions for performing a second alignment of the flexible substrate comprise:

one or more instructions for translating the flexible substrate in at least one dimension to position a chamber in preparation for capturing the second image.

48. The one or more computer-readable media of claim 40 wherein a volume of the fluid is less than a nanoliter.

49. The one or more computer-readable media of claim 40 wherein the first additional fiducial marking is a company logo.

50. The one or more computer-readable media of claim 40 wherein the at least three fiducial markings comprise a company logo.

51. The one or more computer-readable media of claim 40 wherein the flexible substrate is deformable in three dimensions.

52. The one or more computer-readable media of claim 51 wherein the flexible substrate is deformed by actions selected from the group consisting of fabrication, handling, and protocols.

53. The one or more computer-readable media of claim 52 wherein the protocols can result in the flexible substrate swelling or contracting.

54. The one or more computer-readable media of claim 51 wherein a relationship between the design space and the measurement space is non-planar.

55. The one or more computer-readable media of claim 54 wherein the flexible substrate is deformed such that a planar transformation is capable to approximately determine an actual location of the first chamber.

56. The one or more computer-readable media of claim 40, and further comprising:

one or more instructions for normalizing the second image;

one or more instructions for white balancing the second image;

one or more instructions for converting the second image from a first image depth to a second image depth.

57. The one or more computer-readable media of claim 56 wherein the first image depth is 16 bits and the second image depth is 8 bits.

58. The one or more computer-readable media of claim 40 wherein the transformation between the design space and the measurement space is non-planar.

59. The one or more computer-readable media of claim 58 wherein the non-planar transformation comprises a three dimensional parabolic mapping.

60. The one or more computer-readable media of claim 58 wherein the non-planar transformation is updated using information obtained by characterization of a second additional fiducial marking.

84. The one or more computer-readable media of claim 41 wherein the at least three actual locations are determined sequentially.

61. A method for processing a microfluidic device, the method comprising:

providing a flexible substrate comprising one or more well regions and a plurality of fiducial marks, the well regions being capable of holding a fluid therein, at least three of the fiducial marks being within a vicinity of one of the well regions;

locating the flexible substrate on a stage;

capturing an image of at least the three fiducial marks within the vicinity of the one well region of the flexible substrate, wherein in a first state the at least three fiducial marks correspond to at least three design locations respectively, the at least three design locations having a first spatial relationship with respect to each other and being associated with a design space, and in a second state one or more actual locations of the three fiducial marks are shifted from the at least three design locations such that the at least three fiducial marks have a second spatial relationship with respect to each other different from the first spatial relationship and are associated with a measurement space;

generating a mapping from the design space to the measurement space based on at least the captured image;

aligning the flexible substrate to an image acquisition location using at least the mapping from the design space and at least one additional fiducial mark associated with the one well region;

acquiring a second image of at least the one well region; and

storing the second image in a memory.

62. The method of claim 61 wherein the second image comprises 3900 by 3030 pixels.

63. The method of claim 62 wherein the second image comprises a 16 bit image.

64. The method of claim 61 wherein the memory is a computer memory.

65. The method of claim 61 wherein aligning the flexible substrate comprises acquiring a low-resolution image, normalizing the low-resolution image, and median correcting the low-resolution image.

66. The method of claim 65 wherein aligning the flexible substrate further comprises segmenting the image, performing blob analysis, and translating the flexible substrate in at least one dimension.

67. The method of claim 66 wherein translating the flexible substrate in at least one dimension comprises translating the stage to position a metering cell in preparation for capturing a high-resolution image.

68. The method of claim 61 wherein a volume of the fluid is less than a nanoliter.

69. The method of claim 61 wherein the at least one additional fiducial mark is a company logo.

70. The method of claim 61 wherein the at least three fiducial marks are alignment marks.

71. The method of claim 70 wherein the alignment marks are portions of the wells.

72. The method of claim 61 wherein the flexible substrate is deformable in three dimensions.

73. The method of claim 72 wherein the flexible substrate is deformed by actions selected from the group consisting of fabrication, handling, and protocols.

74. The method of claim 73 wherein the protocols can result in the flexible substrate swelling or contracting.

75. The method of claim 74 wherein the flexible substrate is deformed such that there is no linear mapping between the design space and the measurement space.

76. The method of claim 75 wherein the flexible substrate is deformed such that a planar mapping is insufficient to accurately predict the image acquisition location.

77. The method of claim 61 wherein prior to storing the second image in a memory, the second image is normalized, white balanced, and converted to a reduced image depth.

78. The method of claim 77 wherein the reduced image depth is reduced from a 16 bit image to an 8 bit image.

79. The method of claim 61 further comprising creating a higher order mapping from a design space to a measurement space based on at least the mapping from the design space and the at least one additional fiducial mark.

80. The method of claim 79 wherein the higher order mapping comprises a three dimensional parabolic mapping.

81. The method of claim 79 further comprising updating the higher order mapping using information obtained by characterization of a second additional fiducial mark.

82. A system for processing one or more microfluidic devices, the system including one or more computer memories, the system also including a stage for locating a flexible substrate and an imaging system operable to acquire images of the flexible substrate, the flexible substrate comprising one or more well regions and a plurality of fiducial marks, the well regions being capable of holding a fluid therein, at least three of the fiducial marks being within a vicinity of one of the well regions, the one or more computer memories comprising one or more computer codes, the one or more computer codes including:

a first code directed to capturing an image using the imaging system of at least the three fiducial marks within the vicinity of the one well region of the flexible substrate, wherein in a first state the at least three fiducial marks correspond to at least three design locations respectively, the at least three design locations having a first spatial relationship with respect to each other and being associated with a design space, and in a second state actual locations of one or more of the at least three fiducial marks are shifted from the at least three design locations such that the at least three fiducial marks have a second spatial relationship with respect to each other different from the first spatial relationship and are associated with a measurement space;

a second code directed to generating a mapping from the design space to the measurement space based on the captured image;

a third code directed to aligning the flexible substrate to an image acquisition location using at least the mapping from the design space and at least one additional fiducial mark associated with the one well region;

a fourth code directed to acquiring a high-resolution image of at least the one well region using the imaging system; and

a fifth code directed to storing the high-resolution image in a memory.