METHODS, SYSTEMS AND APPARATUS FOR PERFORMING FLOW CYTOMETRY

• US 20140030696A1
• Filed: 02/15/2012
• Published: 01/30/2014
• Est. Priority Date: 02/15/2011
• Status: Active Grant

• Current Assignee: Microbix Biosystems Incorporated, Lathrop Engineering, Inc. (Tecan Group AG)
• Original Assignee: Microbix Biosystems Incorporated
• Inventors: Luscher, Mark, Marks, Randall
• Granted Patent: US 9,556,416 B2
• Time in Patent Office: Days
• US Class Current: 435/3
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/1436 the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 33/487 of liquid biological material
• CPC Class Codes: G01N 33/4915 using flow cells flow cytometry G01N15/14

Abstract

An apparatus for detecting an analyte in a sample includes an illumination source for generating electromagnetic energy to illuminate the sample at an interrogation region, a concave collector element having an optical axis, and a focal point, the interrogation region being coincident with the focal point of the concave collector element, a closed flow cell having a flow path defined between a sample inlet and a sample outlet, the flow path passing through the interrogation region and a sorting region disposed downstream of the interrogation region. The portion of the flow path passing through the interrogation region is coaxially aligned with the optical axis of the concave collector element. The apparatus further includes the sample comprising or suspected of comprising the analyte and flowing in the flow path, wherein the analyte generates a detectable signal in response to illumination. The apparatus also includes a detector for detecting the detectable signal.

Claims

1. An apparatus for detecting an analyte in a fluidic sample, comprising:

an illumination source for generating electromagnetic energy to illuminate the sample at an interrogation region;

a concave collector element comprising a vertex, an optical axis, and a focal point, the interrogation region being coincident with the focal point of the concave collector element;

a closed flow cell comprising;

a sample inlet,

a sample outlet,

a flow path defined between the sample inlet and sample outlet, the flow path passing through the interrogation region and a sorting region disposed downstream of the interrogation region, wherein the portion of the flow path passing through the interrogation region is coaxially aligned with the optical axis of the concave collector element, and wherein at least the portion of the flow path passing through the interrogation region and the sorting region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation, and

the sample comprising or suspected of comprising the analyte and flowing in the flow path, wherein;

the sample flows as a core-in-sheath flow or laminar flow at least through the interrogation region,

the sample has a direction of flow moving from the interrogation region towards the vertex of the collector element, or a direction of flow moving from the vertex of the collector towards the interrogation region and a portion of the flow path passes through an internal volume of the collector element, and

the analyte generates a detectable signal in response to illumination; and

a detector for detecting the detectable signal.

7. The apparatus of claim 1, wherein the illumination source illuminates the sample from one or more directions, from angles of arc greater than 0 degrees and less than or equal to 360 degrees over a plane substantially orthogonal to the flow axis at the interrogation region.

8. The apparatus of claim 1, wherein the illumination source illuminates the sample from one or more directions, from angles of arc greater than 0 degrees and less than or equal to 360 degrees over a plane at an angle oblique to the flow axis at the interrogation region.

9. The apparatus of claim 1, wherein the illumination source illuminates the sample from one or more directions over points, arcs, angles, solid angles, or regions of arbitrary shape on the surface of a virtual sphere surrounding the interrogation region.

10. The apparatus of claim 1, wherein the illumination source illuminates the sample with substantial rotational symmetry about the axis of flow at the interrogation region, exclusive of interruptions by obstructions in the path of the illumination light.

11. The apparatus of claim 1, further comprising:

a conical optical element, wherein the conical optical element receives the electromagnetic energy from the illumination source; and

a concave optical element comprising a focal point coincident with the interrogation region, wherein the concave optical element reflects electromagnetic energy from the conical element and focuses the electromagnetic energy on the interrogation region to illuminate the sample.

12. The apparatus of claim 1, further comprising:

a conical optical element, wherein the conical optical element receives the electromagnetic energy from the illumination source; and

a ring-shaped optical element arranged relative to the conical optical element to collect electromagnetic energy from the conical element and focus the electromagnetic energy on the interrogation region to illuminate the sample.

15. The apparatus of claim 1, further comprising first and second concave optical elements arranged such that the first concave optical element receives the electromagnetic energy from the illumination source and reflects an annular beam of electromagnetic energy to the second concave optical element, and the second concave optical element focuses the annular beam of electromagnetic energy on the interrogation region to illuminate the sample.

16. The apparatus of claim 15, wherein the first and second concave optical elements each comprise an optical axis and the optical axes are coaxially aligned.

17. The apparatus of claim 16, wherein the optical axes of the first and second concave optical elements are coaxially aligned with the optical axis of the collector element, and the second concave optical element focuses the annular beam of electromagnetic energy on the interrogation region axisymmetrically about the interrogation region.

23. The apparatus of claim 1, wherein the flow cell further comprises a spherical element surrounding the interrogation region, the spherical element having an index of refraction that is within 0.1 greater than or less than an index of refraction of the flow cell wall.

2-6. -6. (canceled)

13-14. -14. (canceled)

18-22. -22. (canceled)

24. An apparatus for detecting an analyte in a fluidic sample, comprising:

an illumination source for illuminating a sample at an interrogation region;

first and second parabolic optical elements each comprising an optical axis and a focal point;

a conical optical element disposed within an internal volume of the first parabolic optical element, wherein the illumination source is focused on the conical optical element;

an ellipsoidal collector element comprising a vertex, an optical axis, and a focal point, wherein the first and second parabolic optical elements, the conical optical element, and the ellipsoidal collector element are coaxially aligned, and the interrogation region is coincident with the focal point of the ellipsoidal collector element;

a closed flow cell comprising;

a sample inlet,

a sample outlet,

a flow path defined between the sample inlet and sample outlet, the flow path passing through the interrogation region and a sorting region disposed downstream of the interrogation region, wherein the portion of the flow path passing through the interrogation region is coaxially aligned with the optical axis of the concave collector element, and wherein at least the portion of the flow path passing through the interrogation region and the sorting region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation,

a spherical element surrounding the interrogation region, the spherical element having an index of refraction that is within 0.1 greater than or less than the index of refraction of the flow cell wall at the interrogation region;

the sample comprising or suspected of comprising the analyte and flowing in the flow path, wherein;

the sample flows as a core-in-sheath flow or laminar flow at least through the interrogation region,

the second parabolic optical element focuses the electromagnetic energy axisymmetrically on the sample at the interrogation region, and

the analyte generates a detectable signal in response to illumination; and

a detector for detecting the detectable single, the detectable signal being collected by the ellipsoidal collector element and reflected to the detector.

32. The apparatus of claim 24, wherein the analyte comprises first and second analytes, the apparatus further comprising a logic routine operating on a processor communicatively coupled to the detector, the logic routine operable to (1) determine from the detectable signal whether the analyte from which the detectable signal was generated is a first analyte or a second analyte, and (2) output a signal based on the determination.

33. The apparatus of claim 32, further comprising a controllable sorting energy source communicatively coupled to the logic routine and operable to selectively sort at the sorting region either the first or second analyte according to the output signal from the logic routine.

25-31. -31. (canceled)

34. (canceled)

35. A method of detecting an analyte in a fluidic sample using an apparatus comprising an illumination source, a concave collector element having a vertex, an optical axis, and a focal point, and an interrogation region coincident with the focal point of the concave collector element, the method comprising:

controlling a flow of a sample in a closed flow cell to have a direction of flow (1) from the vertex of the collector element towards the interrogation region, or (2) from the interrogation region towards the vertex of the collector element, the flow cell comprising;

a sample inlet,

a sample outlet, and

a flow path defined between the sample inlet and sample outlet, the flow path passing through the interrogation region and a sorting region disposed downstream of the interrogation region, wherein the portion of the flow path passing through the interrogation region is coaxially aligned with the optical axis of the concave collector element, and wherein at least the portion of the flow path passing through the interrogation region and the sorting region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation,

wherein a portion of the flow path passes through the collector element when the direction of flow is from the interrogation region towards the vertex of the collector element;

illuminating the sample at the interrogation region, wherein the analyte generates a detectable signal in response to illumination;

collecting the detectable signal with the concave collector element, wherein the collector element reflects the detectable signal to the detector; and

detecting the detectable signal with the detector.

36. The method of claim 35, wherein the closed flow cell further comprises a spherical element surrounding the interrogation region, the spherical element having an index of refraction that is within 0.1 greater than or less than an index of refraction of the flow cell wall.

42. The method of claim 35, wherein illuminating the sample comprises:

reflecting electromagnetic energy from the illumination source to a concave optical element using a conical optical element; and

focusing the electromagnetic energy on the interrogation region using the concave optical element, the concave optical element comprising a focal point coincident with the interrogation region.

43. The method of claim 35, wherein illuminating the sample comprises:

reflecting electromagnetic energy from the illumination source to a ring-shaped optical element using a conical optical element, wherein the conical optical element is arranged relative to the ring-shaped optical element such that the electromagnetic energy is received by the ring-shaped optical element and

focusing the electromagnetic energy on the interrogation region using the ring-shaped optical element.

46. The method of claim 35, wherein illuminating the sample comprises reflecting electromagnetic energy from the illumination source as an annular beam to a second concave optical element using a first concave optical element;

and

focusing the annular beam of electromagnetic energy on the interrogation region using the second concave optical element.

47. The method of claim 46, wherein the first and second concave optical elements each comprise an optical axis and the optical axes are coaxially aligned.

48. The method of claim 47, wherein the optical axes of the first and second concave optical elements are coaxially aligned with the optical axis of the collector element.

49. The method of claim 35, wherein the direction of flow of the sample is from the vertex of the collector element towards the interrogation region, and a portion of the flow path passes through the collector element.

37-41. -41. (canceled)

44-45. -45. (canceled)

50-53. -53. (canceled)

54. A method of detecting an analyte in an apparatus comprising an illumination source, a conical optical element, first and second parabolic optical elements, an ellipsoidal collector element having a vertex, an optical axis and a focal point, and an interrogation region defined at the focal point of the ellipsoidal collector element, the method comprising:

controlling a flow of a sample comprising or suspected of comprising an analyte in a closed flow cell comprising;

a sample inlet,

a sample outlet,

a flow path defined between the sample inlet and sample outlet, the flow path passing through the collector element, the interrogation region, and a sorting region disposed downstream of the interrogation region, wherein the portion of the flow path passing through the interrogation region is coaxially aligned with the optical axis of the elliptical collector element, and wherein at least the portion of the flow path passing through the interrogation region and the sorting region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation, and

a spherical element surrounding the interrogation region, the spherical element having an index of refraction that is that is within 0.1 greater than or less than an index of refraction of the flow cell wall;

illuminating the analyte at the interrogation region using an optical arrangement, wherein the analyte generates a detectable signal in response to illumination and the optical arrangement comprises;

the first and second parabolic optical elements each comprising an optical axis and a focal point, and

the conical optical element disposed within an internal volume of the first parabolic optical element, wherein the conical optical element reflects electromagnetic energy from an illumination source to the first parabolic optical element, the first parabolic optical element reflects the electromagnetic energy to the second parabolic optical element, and the second parabolic optical element focuses the electromagnetic energy on the interrogation region;

collecting the detectable signal with the ellipsoidal collector element, wherein the ellipsoidal collector element reflects the detectable signal to a detector; and

detecting the detectable signal with the detector.

60. The method of claim 54, wherein the flow path is coaxially aligned with the optical axis of the collector element.

61. The method of claim 54, wherein the flow cell is a capillary tube.

63. The method of claim 54, wherein the analyte comprises first and second analytes, the method further comprising:

receiving data from the detector after detecting the detectable signal;

determining from the received data the presence in the interrogation region of the analyte; and

determining from the received data whether the analyte is the first or second analyte.

64. The method of claim 63, further comprising a controlling a sorting energy source to selectively sort at the sorting region one of the first or second analyte based on the determination of the analyte being the first or second analyte.

65. The method of claim 64, wherein selective sorting comprises derivatizing, killing, damaging, modifying, disrupting, or fragmenting the first or second analyte.

66. The method of claim 54, further comprising:

receiving data from the detector after detecting the detectable signal, and

controlling a sorting energy source to destructively energize the analyte at the sorting region based on the received data.

67. The method of claim 66, wherein energizing the analyte comprises derivatizing, killing, damaging, modifying, disrupting, or fragmenting the analyte.

55-59. -59. (canceled)

62. (canceled)

68-80. -80. (canceled)