Flow cell alignment methods and systems

• US 9,591,268 B2
• Filed: 03/15/2013
• Issued: 03/07/2017
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Original Assignee: Qiagen Waltham, Inc.
• Inventors: Stoops, David Ray, Veatch, Phillip Alan, Gordon, Steven Jeffrey
• Primary Examiner(s): Czekaj, Dave
• Assistant Examiner(s): SLATER, ALISON T
• Application Number:
  US13/832,509
• Publication Number:
  US 20140267669A1
• Time in Patent Office: 1,453 Days
• Field of Search: 348/79, 382/128
• US Class Current: 1/1
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 2015/0038 Investigating nanoparticles
• CPC Class Codes: G01N 2015/144 Imaging characterised by its optical setup
• CPC Class Codes: G02B 21/10 affording dark-field illumination G02B21/14 and G02B21/125 take precedence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence
• CPC Class Codes: G06T 2200/32 involving image mosaicing
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/73 using feature-based methods
• CPC Class Codes: H04N 7/18 Closed-circuit television [CCTV] systems, i.e. systems in which the video signal is not broadcast

Abstract

Imagers and alignment methods for use by imagers imaging deoxyribonucleic acid (DNA) fragments on a flow cell are disclosed. The imagers capture intensity values at DNA fragment bead locations in tiles with each tile having a reference location in the flow cell. Flow cells may be aligned by obtaining a dark field image of each tile during a first imaging session, identifying dark field constellations of bead locations within two separate tiles during the first imaging session, identifying corresponding constellations during a second imaging session, altering the reference location of at least one tile during the second imaging session to correct for a linear offset in the corresponding constellations, and applying at least one correction factor for reading out intensity values from the imager for the bead locations in the flow cell to correct for an angular offset determined from offsets in the corresponding constellations.

Claims

1. An alignment method for use by an imager imaging deoxyribonucleic acid (DNA) fragments on a flow cell, the imager capturing intensity values at DNA fragment bead locations in tiles with each tile having a reference location in the flow cell, the method comprising:

during a first imaging session;

positioning the flow cell on an xy stage with the flow cell facing an imaging axis, the xy stage being movable along an x-axis and a y-axis, the x-axis being perpendicular to the y-axis, and the imaging axis being perpendicular to the x-axis and the y-axis,

illuminating the flow cell from the side of the flow cell with a first light to cause the first light to strike the DNA fragment beads from above the xy stage and at an angle relative to the imaging axis, and reflect off the DNA fragment bead locations,

operating the xy stage to reposition the flow cell to obtain a respective first image of each of a first tile and a second tile during a first imaging session, the respective first image comprising reflections of the first light, and

identifying a first reference constellation of bead locations within the first tile and a second reference constellation of bead locations within the second tile during the first imaging session; and

during a second imaging session;

identifying a first pattern of light corresponding to the first reference constellation in the first tile, and a second pattern of light corresponding to the second reference constellation in the second tile,

determining a first offset between the first reference constellation during the first imaging session and the first pattern of light during the second imaging session,

determining a second offset between the second reference constellation during the first imaging session and the second pattern of light during the second imaging session,

moving the xy stage to alter the reference location of at least one tile during the second imaging session to correct for the first offset, and

applying at least one correction factor for reading out intensity values from the imager for the bead locations in the flow cell to correct for an angular offset determined from the first offset and the second offset.

2. The method of claim 1, where in the first offset comprises a linear offset of the flow cell between the first imaging session and the second imaging session.

3. The method of claim 1, further comprising:

obtaining a fluorescent field image for each tile of the flow cell during the second imaging session;

reading out the intensity values of the fluorescent field image at the bead locations identified in the first image as corrected by the applied at least one correction factor.

4. The method of claim 3, wherein the step of obtaining a fluorescent field image for each tile of the flow cell comprises:

positioning the flow cell for fluorescent imaging based on the altered reference location of the tile;

illuminating the tile with light of a first color;

capturing light emitted from the tile;

determining at least one additional correction factor by matching a portion of the emitted light to a corresponding portion in the first image;

applying the at least one additional correction factor for reading out intensity values;

wherein the intensity values are read out as corrected by the at least one correction factor and the at least one additional correction factor.

5. The method of claim 4, wherein the step of obtaining a fluorescent field image for each tile further comprises:

illuminating the tile with light of a second color;

capturing light emitted from the tile;

determining at least one additional correction factor by matching a portion of the emitted light to a corresponding portion in the first image;

applying the at least one additional correction factor for reading out intensity values;

wherein the intensity values associated with illuminating the tile with light of a second color are read out as corrected by the at least one correction factor and the at least one additional correction factor.

6. The method of claim 1, wherein the flow cell is imaged in 72 tiles including 3 rows with 24 tiles in each row, the first and second tiles are in the center row, and the second tile is at least 5 tiles away from the first tile.

7. The method of claim 6, wherein the second tile is at least 9 tiles away from the first tile.

8. The method of claim 1, further comprising:

removing the flow cell from the xy stage after the first imaging session; and

repositioning the flow cell on the xy stage during the second imaging session.

9. The method of claim 1, wherein the at least one correction factor comprises a difference between the first offset and the second offset.

10. The method of claim 8, further comprising:

obtaining a fluorescent field image for each tile of the flow cell during the second imaging session;

reading out the intensity values of the fluorescent field image at the bead locations identified in the first image as corrected by the applied at least one correction factor.

11. The method of claim 10, wherein the step of obtaining a fluorescent field image for each tile of the flow cell comprises:

positioning the flow cell for fluorescent imaging based on the altered reference location of the tile;

illuminating the tile with light of a first color;

capturing light emitted from the tile;

determining at least one additional correction factor by matching a portion of the emitted light to a corresponding portion of the first image;

applying the at least one additional correction factor for reading out intensity values;

wherein the intensity values are read out as corrected by the at least on correction factor and the at least one additional correction factor.

29. The method of claim 1, wherein the first light source comprises a dark field light emitting diode.

12. An imager for imaging deoxyribonucleic acid (DNA) fragments on a flow cell, the imager capturing intensity values at DNA fragment bead locations in tiles with each tile having a reference location in the flow cell, the imager comprising:

an xy stage supporting the flow cell with the flow cell facing an imaging axis, the xy stage being movable along an x-axis and a y-axis, the x-axis being perpendicular to the y-axis, and the imaging axis being perpendicular to the x-axis and the y-axis;

a dark field light emitting diode positioned along the y-axis to one side of the flow cell, the dark field light emitting diode being configured to illuminate the flow cell with a first light that strikes the flow cell from above the xy stage and a direction that is angled relative to the imaging axis;

a camera configured to capture images of flow cell tiles; and

a processor coupled to the xy stage and the camera, the processor configured to position the xy stage at a plurality of tile locations along the x-axis and the y-axis, and control the camera to;

reposition the xy stage to obtain a first image of each tile during a first imaging session, the first image comprising reflections from the first dark field LED;

identify a first reference constellation of bead locations within a first tile and a second reference constellation of bead locations within a second tile during the first imaging session;

identify, during a second imaging session, a first pattern of light corresponding to the first reference constellation and a second pattern of light corresponding to the second reference constellation;

determine a first offset between the first reference constellation during the first imaging session and the first pattern of light during the second imaging session;

determine a second offset between the second reference constellation during the first imaging session and the second pattern of light during the second imaging session;

alter the reference location of at least one tile during the second imaging session when positioning the xy stage to correct for the first offset; and

apply at least one correction factor for reading out intensity values from the camera for the bead locations in the flow cell to correct for an angular offset determined from the first offset and the second offset.

13. An alignment method for use by an imager imaging deoxyribonucleic acid (DNA) fragments in a flow cell, the method comprising:

providing a flow cell chip that extends in a horizontal plane having an imaging axis extending perpendicular thereto, the flow cell chip having a flow cell comprising a plurality of tiles, wherein each tile comprises a respective reference location and a respective plurality of DNA fragment beads;

positioning the flow cell chip on an xy stage, the xy stage being movable along an x-axis and a y-axis the x-axis being perpendicular to the y-axis and the imaging axis being perpendicular to the x-axis and the y-axis

during a first imaging session;

directing a first light from a side of the flow cell chip in relation to the imaging axis, such that the first light strikes the flow cell chip from above the xy stage and at an angle relative to the imaging axis, to thereby illuminate the flow cell chip from the side,

positioning the flow cell chip with a reference location of a first tile at a predetermined position in relation to a camera, and obtaining an image of reflections along the imaging axis of the first light from DNA fragment beads in the first tile with each reflection indicating the position of a respective DNA fragment bead, and

identifying a group of the reflections of the first light from the DNA fragment beads in the first tile as a first reference constellation;

after the first imaging session, repositioning the flow cell chip in relation to the camera;

during a second imaging session;

attempting to position the flow cell chip with the reference location of the first tile at the predetermined position in relation to the camera, and

examining DNA fragment bead locations in the first tile to determine the location of the first reference constellation; and

comparing the location of the first reference constellation during the second imaging session to the location of the first reference constellation during the first imaging session to determine a first offset between the position of the first tile in relation to the camera during the first imaging session and the position of the first tile in relation to the camera during the second imaging session.

14. The method of claim 13, wherein the first offset comprises at least one of a linear offset in the horizontal plane and an angular offset about the imaging axis.

15. The method of claim 13, wherein the respective reference location of each tile comprises a geometric center of each tile.

16. The method of claim 13, wherein the first reference constellation is located in a portion of the first tile surrounding a geometric center of the first tile.

17. The method of claim 13, wherein the first light comprises a red light emitting diode.

18. The method of claim 13, further comprising obtaining a fluorescent field image of at least one tile by:

activating a second light to cause the respective DNA fragment beads in the at least one tile to emit light at a wavelength that is different from a wavelength of the second light; and

obtaining an image of the light emitted from the respective DNA fragment beads in the at least one tile.

19. The method of claim 18, wherein the step of activating a second light is preceded by:

generating a modified respective reference location of at least one tile by subtracting the first offset from the respective reference location of the at least one tile; and

positioning the flow cell chip with the modified respective reference location of the at least one tile at the predetermined position in relation to the camera.

20. The method of claim 18, further comprising applying a correction factor to the fluorescent field image to remove a positional error caused by the first offset.

21. The method of claim 20, wherein applying the correction factor comprises determining intensity values of the fluorescent field image at respective intensity locations and applying a correction factor based on the first offset to shift the respective intensity locations to correspond to positions of respective DNA fragment beads identified during the first imaging step.

22. The method of claim 18, wherein:

activating a second light to cause the respective DNA fragment beads in the at least one tile to emit light at a wavelength that is different from a wavelength of the second light comprises positioning a mirror in an optical path from the at least one tile to the camera; and

activating a first light located to a side of the flow cell chip in relation to the imaging axis, to thereby illuminate the flow cell chip from the side comprises removing the mirror from the optical path from the at least one tile to the camera.

23. The method of claim 13, further comprising:

during the first imaging session;

positioning the flow cell chip with a reference location of a second tile at the predetermined position in relation to the camera, and obtaining an image of reflections of the first light from DNA fragment beads in the second tile, and

identifying a group of the reflections of the first light from the DNA fragment beads in the second tile as a second reference constellation;

during the second imaging session;

attempting to position the flow cell chip with the reference location of the second tile at the predetermined position in relation to the camera, and

examining DNA fragment bead locations in the second tile to determine the location of the second reference constellation; and

comparing the location of the second reference constellation during the second imaging session to the location of the second reference constellation during the first imaging session to determine a second offset between the position of the second tile in relation to the camera during the first imaging session and the position of the second tile in relation to the camera during the second imaging session.

24. The method of claim 23, wherein the first offset comprises a linear offset in the horizontal plane, and the second offset comprises an angular offset about the imaging axis.

25. The method of claim 24, further comprising:

generating a modified respective reference location of at least one tile by subtracting the first offset from the respective reference location of the at least one tile;

positioning the flow cell chip with the modified respective reference location of the at least one tile at the predetermined position in relation to the camera;

obtaining a fluorescent field image of the least one tile by;

directing a second light to strike the flow cell chip along the imaging axis to cause the respective DNA fragment beads in the at least one tile to emit light at a wavelength that is different from a wavelength of the second light, and

obtaining an image of the light emitted along the imaging axis from the respective DNA fragment beads in the at least one tile; and

applying a correction factor to the fluorescent field image to remove a positional error caused by the second offset.

26. The method of claim 23, wherein the first light illuminates the first tile and the second tile at the same angle of incidence.

27. The method of claim 13, wherein examining DNA fragment bead locations in the first tile to determine the location of the first reference constellation during the second imaging session comprises:

directing the first light from the side of the flow cell chip in relation to the imaging axis, such that the first light strikes the flow cell chip from above the xy stage and at the angle relative to the imaging axis, to thereby illuminate the flow cell chip from the side;

obtaining an image of reflections of the first light from DNA fragment beads in the first tile; and

identifying the group of the reflections of the first light from the DNA fragment beads in the first tile as the first reference constellation.

28. The method of claim 13, wherein examining DNA fragment bead locations in the first tile to determine the location of the first reference constellation during the second imaging session comprises:

directing a second light to cause the DNA fragment beads in the first tile to emit light at a wavelength that is different from a wavelength of the second light;

obtaining an image of the light emitted along the imaging axis from the DNA fragment beads in the first tile; and

identifying a portion of the light emitted from the DNA fragment beads in the first tile as the first reference constellation.