Methods, systems, and apparatus for performing flow cytometry
An apparatus for identifying analytes of a first type in a fluid sample having a plurality of types includes a closed flow cell. The flow cell includes a sample inlet, a sample outlet. A flow path, between the inlet and outlet, passing through an interrogation region and at least a portion extending from immediately upstream to immediately downstream of the interrogation region is bounded by a flow cell well to form a continuous closed flow path. The apparatus also includes a spherical element having a center point coincident with the interrogation region. A hydrodynamic focusing element upstream of the interrogation region creates a core-in-sheath flow along a flow axis of the flow path as it passes through the interrogation region. Further, the apparatus includes a concave, ellipsoidal collector element having a first focal point disposed in the interrogation region of the flow path. A plurality of beams of electromagnetic energy converge non-axisymmetrically at the interrogation region, each of the plurality of beams orthogonal to the flow axis.
1. An apparatus for identifying analytes of a first type in a fluid sample comprising analytes of a plurality of types, 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, wherein at least the portion of the flow path extending from immediately upstream of the interrogation region to immediately downstream of the interrogation region is bounded by a flow cell wall to form a continuous closed flow path, without division or bifurcation;
a spherical molding surrounding the flow path and having a center point coincident with the interrogation region such that electromagnetic energy propagating from the interrogation region to an exterior surface of the spherical molding intersects the exterior surface at an angle orthogonal to the exterior surface;
a hydrodynamic focusing element upstream of the interrogation region and fluidically coupled with the flow path of the flow cell and creating a core-in-sheath flow along a flow axis of the flow path as it passes through the interrogation region, the core-in-sheath flow comprising a core flow carrying the analytes, the hydrodynamic focusing element comprising (i) an outer flow path configured for fluidic coupling to a source of sheath fluid and having a sheath fluid flow axis and (ii) an inner flow path, disposed within the outer flow path, configured for fluidic coupling to a source of the fluid sample, wherein the sample flow axis is generally coaxial with the sheath fluid flow axis, and (iii) a wall surrounding the inner flow path upstream of the interrogation region;
a concave, ellipsoidal collector element comprising a vertex, an optical axis, a first focal point disposed in the interrogation region of the flow path, a second focal point, and a reflective surface disposed in an optical path between the first focal point and the second focal point, such that the reflective surface directs a signal from the analyte in the interrogation region to the second focal point;
illumination means comprising a plurality of beams of electromagnetic energy converging non-axisymmetrically at the interrogation region, each of the plurality of beams orthogonal to the flow axis.
2. An apparatus according to claim 1 , wherein the spherical molding, or a 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. An apparatus according to claim 1 , wherein the flow path is divided downstream of a sorting region of the flow path, and configured to separate all or part of the core flow from the sheath flow downstream of the interrogation region.
4. An 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. An apparatus according to claim 1 , further comprising a source of direct, destructive radiation, configured to deliver the radiation to a point in the flow path between the interrogation region and the sample outlet.
6. An apparatus according to claim 5 , wherein the source of direct, destructive radiation comprises a pulsed laser.
7. An apparatus according to claim 1 , wherein the hydrodynamic focusing element is configured to keep an energy dissipation rate (EDR) below a predetermined threshold, the EDR calculated according to the equation ε=μ(∇U+∇UT):=U,
wherein ε is the EDR, μ is the viscosity, (∇U+∇UT) is the stress tensor, ∇U is the velocity gradient tensor, and ∇UT is the transpose of ∇U.
8. An apparatus according to claim 1 , wherein the wall surrounding the inner flow path of the hydrodynamic focusing element has an outer surface tapered at an end disposed toward a point of convergence of the inner and outer flow paths.
9. An apparatus according to claim 1 , wherein the optical axis of the ellipsoidal collector element is coaxial with the flow path as the flow path passes through the interrogation region.