Fluorescence-activated cell detector
An apparatus, and method of use of such apparatus, for detecting particles of interest that are dispersed in a fluid mix, which typically includes other particles. The apparatus includes an interrogation platform arranged to operate in harmony with an opaque member having an orifice sized to promote single-file travel of the particles there-through. A radiation source is disposed on one side of the opaque member, and a radiation detector is disposed on the other side of the opaque member. Particles of interest are tagged using antibody-binding, fluorescing molecules. Radiation from the source causes the tagged particles to fluoresce in the vicinity of, and passing through, the orifice. The resulting fluorescence is detected by the radiation detector and indicates passage of the particles of interest. One workable opaque member is advantageously included in a thin film assembly carried on a removable and disposable card that is adapted for reception in the interrogation platform.
1. An apparatus, comprising:
an interrogation platform disposable in association with a plumbing arrangement adapted to transport particles suspended in a fluid, said plumbing arrangement being configured to urge transit of said particles in substantially single-file through a first orifice disposed to provide a first flow path through a substantially opaque member;
a radiation source disposed on a first side of said opaque member, said radiation source being arranged to apply primary radiation in a direction along a radiation vector into a zone associated with said first orifice effective to excite a first subset of particles passing through said zone operably to cause an emission of fluorescence from a first particle selected from said first subset, with a first portion of fluorescence from said first particle being directed for transmission in a direction from said first side toward a second side of said substantially opaque member and through said first orifice; and
a radiation detector disposed on said second side of said substantially opaque member, said second side being opposite said first side, said radiation detector being operably arranged for reception and detecting of said first portion.
2. The apparatus according to claim 1 , wherein:
said primary radiation has a first characteristic wavelength; and
said fluorescence has a second characteristic wavelength that is different from said first characteristic wavelength.
3. The apparatus according to claim 2 , further comprising:
a first filter disposed between said radiation source and said radiation detector, said first filter being configured and arranged to resist reception of said primary radiation by said radiation detector.
4. The apparatus according to claim 3 , further comprising:
a second filter disposed on said first side, said second filter being configured and arranged to resist transmission there-through of radiation departing from said first characteristic wavelength.
5. The apparatus according to claim 1 , further comprising:
a collecting lens disposed on said second side, said collecting lens being configured and arranged to urge part of said first portion toward a detecting element of said radiation detector.
6. The apparatus according to claim 5 , wherein:
said collecting lens comprises a fiber optic cable.
7. The apparatus according to claim 5 , wherein:
said collecting lens comprises a convex focusing lens.
8. The apparatus according to claim 1 , wherein:
said radiation vector is oriented at an acute angle to a through-axis of said first orifice.
9. The apparatus according to claim 8 , wherein:
said acute angle is between about 15 degrees and about 75 degrees.
10. The apparatus according to claim 1 , wherein:
said plumbing arrangement is configured to urge transit of said particles in substantially single-file through a plurality of orifices, each such orifice being disposed to provide a respective flow path through said substantially opaque member;
said radiation source is arranged to apply primary radiation into a zone associated with said plurality of orifices effective to excite a first subset of particles passing through said zone operably to cause an emission of fluorescence from certain particles selected from said first subset, with fluorescence from said certain particles being directed for transmission in a direction from said first side toward a second side of said substantially opaque member; and
said radiation detector is operably arranged for reception and detecting of said fluorescence.
11. The apparatus according to claim 1 , wherein said plumbing arrangement comprises structure arranged such that:
fluid flow through said first orifice is directed approximately orthogonal to fluid flow in a channel disposed immediately downstream of said first orifice.
12. The apparatus according to claim 1 , wherein said plumbing arrangement comprises structure arranged such that:
fluid flow through said first orifice is directed approximately orthogonal to fluid flow in a channel disposed immediately upstream of said first orifice.
13. The apparatus according to claim 1 , wherein:
said first orifice has a characteristic dimension sized between about 5 microns and about 200 microns.
14. The apparatus according to claim 1 , wherein:
a thickness of said opaque member is between about 10 microns and about 300 microns.
15. The apparatus according to claim 1 , wherein:
said opaque member comprises a membrane carrying an opaque substance as a first coating disposed on one side thereof.
16. The apparatus according to claim 15 , further comprising:
a second opaque layer as a second coating disposed on a side opposite said one side.
17. The apparatus according to claim 15 , further comprising:
a transmission window formed through the thickness of a removable cartridge, said cartridge being adapted to interface with structure of said interrogation platform, and carrying structure adapted to form said plumbing arrangement.
18. A method for detecting particles of interest, comprising the steps of:
a) preparing a sample of particles suspended in a fluid carrier medium by mixing a quantity of said particles with antibody-bound fluorescently labeled molecules;
b) incubating said sample for a period of time sufficient to permit said antibody-bound fluorescently labeled molecules to bind to said particles of interest in said sample;
c) providing an interrogation platform configured to operate on a detection zone disposed in association with an orifice configured to provide a flow path through a substantially opaque member, said orifice being sized sufficiently in agreement with a characteristic size of said particles of interest as to promote substantially single-file travel of said particles of interest there-through, said interrogation platform further comprising a radiation source disposed on one side of said substantially opaque member and a radiation detector disposed on an opposite side of said substantially opaque member;
d) causing a portion of said sample to flow through said detection zone;
e) using said source of radiation to impinge primary radiation, having a first characteristic wavelength, into said detection zone operably to excite said antibody-bound fluorescently labeled molecules to promote emission there-from of secondary radiation, said secondary radiation having a second characteristic wavelength;
f) using said radiation detector to detect said secondary radiation; and
g) causing said portion of said sample to flow away from said detection zone.
19. The method according to claim 18 , wherein:
the incubation of step b) occurs at a temperature between about 20 degrees Celsius and about 39 degrees Celsius.
20. The method according to claim 18 , wherein:
said opaque member is included in a plumbing arrangement comprising a thin film assembly carried on a removable card, said plumbing arrangement causing fluid flow away from said detection zone to occur in an essentially orthogonal direction compared to fluid flow through said orifice, said removable card being configured and arranged to interface with structure of said interrogation platform; and
step c) further includes inserting said card into operable position in association with said interrogation platform.