Methods, systems and apparatus for performing flow cytometry
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.
1. An apparatus for selectively enriching an X-chromosome bearing or a Y-chromosome bearing population of cells in a fluid sample comprising non-human, mammalian sperm cells, the apparatus comprising:
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 an interrogation region and a sorting region disposed downstream of the interrogation region, wherein at least the portion of the flow path extending from immediately upstream of the interrogation region to immediately downstream of the sorting region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation;
a spherical element having a center point coincident with the interrogation region;
a hydrodynamic focusing element upstream of the interrogation region and creating a core-in-sheath flow along a flow axis of the flow path between the interrogation region and the sorting region, the core-in-sheath flow comprising a core flow carrying the non-human, mammalian sperm cells, the hydrodynamic focusing element configured to maintain below a threshold value a maximum local energy dissipation rate (EDR) experienced by the sperm cells;
a concave, ellipsoidal collector element comprising a vertex, an optical axis, a first focal point disposed in the interrogation region of the flow path, and a second focal point;
illumination means comprising a plurality of beams of electromagnetic energy converging non-axissymetrically at the interrogation region, each of the plurality of beams orthogonal to the flow axis; and
a source of direct, destructive radiation configured to deliver electromagnetic energy to the core flow as the core flow passes through the sorting region.
2. The apparatus according to claim 1 , wherein the spherical element, or portion thereof, is formed of a material having an index of refraction that is within 0.1 greater than or less than an index of refraction of the flow cell wall.
3. The apparatus according to claim 1 , wherein the flow path is configured to separate all or part of the core flow from the sheath flow downstream of the sorting region.
4. The apparatus according to claim 1 , wherein the flow path comprises, at an end proximal to the sample outlet, an enlarged diameter to reduce the velocity of the fluid sample.
5. The apparatus according to claim 1 , wherein the source of direct, destructive radiation comprises a pulsed laser.
6. The apparatus according to claim 1 , wherein each of the plurality of beams of electromagnetic energy converging at the interrogation region is circularly polarized.
7. A method for selectively enriching an X-chromosome bearing or a Y-chromosome bearing population of cells in a fluid sample comprising non-human, mammalian sperm cells, the method comprising:
forming a core-in-sheath flow in a closed 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 an interrogation region and a sorting region disposed downstream of the interrogation region, the flow cell comprising a spherical element having a center point coincident with the interrogation region, wherein at least the portion of the flow path extending from immediately upstream of the interrogation region to immediately downstream of the sorting region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation, the core-in-sheath flow formed by a hydrodynamic focusing element upstream of the interrogation region and creating a core-in-sheath flow along a flow axis of the flow path between the interrogation region and the sorting region, the core-in-sheath flow comprising a core flow carrying the non-human, mammalian sperm cells, the hydrodynamic focusing element configured to maintain below a threshold value a maximum local energy dissipation rate (EDR) experienced by the sperm cells;
configuring a plurality of beams of electromagnetic energy to converge non-axissymetrically at the interrogation region, each of the plurality of beams orthogonal to the flow axis;
collecting a signal received from each of the population of cells passing through the interrogation region, the signal generated as a result of each cell being illuminated by the plurality of beams of electromagnetic energy as the cell passes through the interrogation region, the signal collected by a concave, ellipsoidal collector element comprising a vertex, an optical axis a first focal point disposed in the interrogation region of the flow path, and a second focal point to which the energy collected from the first focal point is transmitted;
detecting the collected signal at a detector;
determining from the detected signal whether each cell is a X-chromosome bearing cell or a Y-chromosome bearing cell;
selectively delivering direct, destructive electromagnetic energy to the core flow as the core flow passes through the sorting region, to enrich the X-chromosome bearing or the Y-chromosome bearing population of cells in the fluid sample.
8. The method according to claim 7 , further comprising separating the core flow from all or part of the sheath flow downstream of the sorting region.
9. The method according to claim 7 , wherein the flow path comprises a portion, downstream of the sorting region, having an enlarged diameter and the method further comprising reducing the velocity of the fluid sample by passing the sample through the portion of the flow path having the enlarged diameter.
10. The method according to claim 7 , wherein selectively delivering direct, destructive electromagnetic energy to the core flow as the core flow passes through the sorting region comprises using a pulsed laser to deliver the direct destructive electromagnetic energy.
11. The method according to claim 7 , wherein configuring the plurality of beams of electromagnetic energy to converge non-axissymetrically at the interrogation region, comprises circularly polarizing each of the plurality of beams of electromagnetic energy.
12. The method according to claim 7 , wherein forming the core-in-sheath flow in a flow cell comprising a spherical element, or portion thereof, surrounding the interrogation region comprises forming the core-in-sheath flow in a flow cell comprising a spherical element formed of a material having an index of refraction that is within 0.1 greater than or less than an index of refraction of the flow cell wall.