Multiple sort flow cytometer

• US 5,483,469 A
• Filed: 08/02/1993
• Issued: 01/09/1996
• Est. Priority Date: 08/02/1993
• Status: Expired due to Term

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Van den Engh, Ger, Esposito, Richard J.
• Primary Examiner(s): Voeltz, Emanuel T.
• Assistant Examiner(s): PIPALA, EDWARD J
• Application Number:
  US08/100,762
• Time in Patent Office: 890 Days
• Field of Search: 364/555, 364/569, 364/413.08, 435/30, 435/7.25, 435/291, 356/72, 356/23, 356/318, 356/336, 436/63, 209/3.1, 324/71.4, 324/71.1
• US Class Current: 702/21
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties

Abstract

A flow cytometer utilizes multiple lasers for excitation and respective fluorescence of identified dyes bonded to specific cells or events to identify and verify multiple events to be sorted from a sheath flow and droplet stream. Once identified, verified and timed in the sheath flow, each event is independently tagged upon separation from the flow by an electrical charge of +60, +120, or +180 volts and passed through oppositely charged deflection plates with ground planes to yield a focused six way deflection of at least six events in a narrow plane.

Claims

1. A Cytometry apparatus for sorting a plurality of cellular events, comprising:

means for providing a sheath flow jet of fluid with cellular matter and events centrally disposed in said sheath flow jet of fluid and for converting said sheath flow jet into a continuous flow of droplets said droplets containing events;

means for obtaining at least a first and second identification parameters of a particular event in said sheath flow jet;

means for plotting each said first identification parameter against said second identification parameter on an orthogonal coordinate domain to obtain a double verification of said event as a specific point of said domain;

means for plotting a plurality of areas on said orthogonal coordinate domain wherein said areas are defined by predetermined boundaries circumventing areas which encompass and delineate particular events;

means for determining within which said defined area each said specific point lies, and thereby determining the identity of said event;

means for designating in which droplet said identified event will reside upon severance from said sheath flow;

means for charging one of a variety of positive and negative electrical potentials to an identified event droplet;

means for shielding said droplet charging process from spurious electric fields; and

means for deflecting and focusing said charged droplets into a plurality of droplet streams confined within a defined plane, wherein each said deflected droplet stream contains only droplets of the same electrical potential and charge, wherein said deflecting and focusing means consists of a pair of oppositely charged plates disposed on each side of said droplet flow and having associated therewith a respective ground plane for each said plate, wherein each said ground plane is curved around each said respective charged plate and is separated from said charged plate by a dielectric to cause a curved and focused electric field between said oppositely charged plates to more accurately focus deflections of said charged droplets.

2. Cytometry apparatus according to claim 1, wherein said means for providing a sheath flow jet and flow of droplets consists of a flow chamber.

3. Cytometry apparatus according to claim 2, wherein said means for obtaining at least a first and second identification parameters consists of at least a first and second laser beams, and a first and second photomultiplier detectors, wherein said laser beams are focused at a first and second points separated in space and time in said sheath flow jet, whereby said laser beams excite designated dye molecules bonded to identified events, whereupon the respective fluorescence emitted by said dye molecules on de-excitation is detected by said first and second detectors.

4. Cytometry apparatus according to claim 3, wherein said means for plotting said event identification parameter points and said event areas, and for determining in which area a particular point lies consists of a computer and logic circuit with a memory of look up tables describing a plurality of event areas.

5. Cytometry apparatus according to claim 4, wherein said means for designating an event droplet consists of a signal processing circuit wherein data from said first detector indicating an event identification parameter passing through said first laser beam is digitized, stored and delayed until a corresponding pulse from said second detector of the same event passing through said second detector is received, and similarly processed to yield a parallel output of both said identification parameters into said computer, wherein transit time of said event and distance between said first and second laser beams can be computed to indicate where said event will be at time of each droplet severance.

6. Cytometry apparatus according to claim 5, wherein said means for charging said event droplets consist of an electrical circuit having a plurality of individual TTL inputs coupling said computer to said flow chamber wherein a high voltage output of said circuit to said flow chamber is determined by said inputs activated by said computer.

7. Cytometry apparatus according to claim 6, wherein said electrical circuit has at least three positive deflection inputs and at least three negative deflection inputs to yield at least three positive deflection outputs and at least three negative deflection outputs.

8. Cytometry apparatus according to claim 7, wherein said means for shielding said droplet charging process consists of a Faraday shield.

9. Cytometry apparatus according to claim 8, wherein said Faraday shield consists of a metallic box surrounding said flow chamber.

10. A method for sorting cellular events, comprising the steps of:

providing a sheath flow of fluid with cellular matter and events suspended within said sheath flow of fluid;

obtaining at least a first and second identification parameters of certain events inside said sheath flow;

plotting said first identification parameters against said second identification parameters to establish a point(s) on an orthogonal coordinate domain, wherein said orthogonal coordinate domain encompasses a plurality of areas, wherein each said area defines a particular event;

identifying said events by determining in which said area each said points lies;

converting said sheath flow into a flow of individual droplets;

identifying in which droplets said identified events reside to designate a specific event droplet;

selecting one of a variety of different positive and negative electrical potentials to tag each said event droplet;

tagging said event droplets by applying an electrical charge to each said event droplet to establish said selected electrical potential on each said identified event droplet thereby converting said flow of droplets into a flow of charged droplets of various different positive and negative electrical potentials;

shielding said droplet charging process from undesirable electric fields; and

passing said flow of charged droplets of various potentials between a pair of oppositely charged plates thereby creating a variety of charged deflected droplets, wherein highly charged droplets are deflected most and lesser charged droplets are deflected least, wherein the step of passing said flow of variously charged event droplets between a pair of oppositely charged plates further utilizes ground planes associated with each said plate to cause a curved and focused electric field between said plates to more accurately focus said deflections of charged particles.

11. The method of claim 10, wherein the step of obtaining said first and second identification parameters consists of focusing a first and second laser beams on said sheath flow and thereby exciting and detecting respective fluorescence of certain dye molecules bonded to particular cellular events.

12. The method of claim 11, wherein the step of identifying specific event droplets consists of computing the flow rate by distance traveled and transit time between said focused laser beams and thereby computing by distance and time yet to travel to said event droplet formation point exactly when said event will be in a droplet.

13. The method according to claim 12, wherein the step for tagging said event droplets, consists of selecting one of at least six different electric potentials and applying a corresponding high voltage to said sheath flow at the exact moment of event droplet severance from said flow.

14. The method according to claim 13, whereby the step of shielding said droplet charging process consists of enclosing said process within a conductive container.

15. A cytometry apparatus, comprising:

a flow chamber issuing a sheath flow jet which is converted to a flow of individual droplets containing fluorescent dye bonded events to be sorted;

a first laser operating at a particular frequency focused at a first point in said sheath flow to irradiate, excite, and thereby cause a particular dye bonded to a specific event to fluoresce;

a first detector issuing a first identification parameter pulse on detection of fluorescence of a particular wavelength from said sheath flow;

at least a second laser operating at said particular frequency and focused at a second point down stream in said sheath flow to irradiate, excite, and thereby cause said particular dye bonded to said specific event to fluoresce again;

at least a second fluorescence detector issuing a second identification parameter pulse on detection of a second fluorescence of said of said particular wavelength from said sheath flow;

a signal processing circuit coupled to each said detector, digitally processing in parallel said identification parameter pulses with respective time delays of said same event pulses;

a logic circuit coupled to said signal processing circuit, containing a look-up table of identified event parameters wherein said table is electronically overlaid on an orthogonal plot of said identification parameters to verify said event identity.

an event multiple charge circuit coupling said logic circuit to said flow chamber applying one of a plurality of different positive and negative electric potential charges to an identified event droplet;

a first deflection plate and a second deflection plate each disposed on opposite sides of said droplet flow, wherein said first deflection plate and said second deflection plate are oppositely charged, wherein said first deflection plate and said second deflection plate each are provided with a ground plane causing a curved electric field between said plates, wherein multiple charged event droplets passing there between are deflected in different paths and focused in a narrow plane between said plates; and

a Faraday shield enclosing said flow chamber.

16. Cytometry multiple sort apparatus delineated in claim 15, wherein said fluorescence detectors are photomultiplier tubes.

17. Cytometry multiple sort apparatus delineated in claim 16, wherein said photo multiplier tubes are provided with spectral filters.

18. Cytometry multiple sort apparatus delineated in claim 17, wherein said spectral filter are preceded by a triple pin-hole barrier enabling a three way split of dye fluorescence anti laser scatter passing therethrough.

19. Cytometry multiple sort apparatus delineated in claim 15, wherein said logic circuit consists of a computer which, upon identification of an event through said look-up table, selects one of a plurality of event droplet charge inputs to said charge circuit.

20. Cytometry multiple sort apparatus delineated in claim 19, wherein said event multiple charge circuit is coupled to said flow chamber and applies a chosen charge to be applied to said flow chamber and attached sheath flow jet at the exact moment an identified event is in droplet formation.

21. Cytometry multiple sort apparatus delineated in claim 20, wherein said multiple charge circuit receives a TTL trigger at one of at least six input selections to yield one of at least six high voltage outputs.

22. Cytometer multiple sort apparatus delineated in claim 21, wherein said high voltage outputs range in six 60 volts steps between -180 volts and +180 volts.