Biological particle sorter

• US 6,482,652 B2
• Filed: 03/21/2001
• Issued: 11/19/2002
• Est. Priority Date: 03/23/2000
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Furlong, Eileen, Profitt, David, Scott, Matthew
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US09/814,345
• Publication Number:
  US 20010036668A1
• Time in Patent Office: 608 Days
• Field of Search: 436/63, 436/164, 436/172, 436/72, 422/73, 209/3.1, 209/552, 209/576, 209/577, 209/579, 209/587, 209/588, 209/906, 356/432, 356/436, 356/440, 356/441, 356/442
• US Class Current: 436/63
• CPC Class Codes: B07C 5/3425 of granular material, e.g. ore particles, grain investigation moving fluids or granular solids using optical means G01N21/85
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: Y10S 209/906 Pneumatic or liquid stream feeding item

Abstract

An automated particle sorter having a fluid flow path, which places single biological particles in an optical cuvette. An exciting light irradiation system having a light source emits a source of light through the cuvette. The light excites a fluorescent substance present on the particle, and the emitted light is detected by a light detection apparatus containing at least two detection elements for measuring the fluorescence emitted from the fluorescent substance. A light separation element separates the fluorescence from the exciting light. A data processor compares the signal received from the fluorescent light; and from the background autofluorescent signal, and according to pre-set parameters, controls the position of a collection conduit between two set points. The first being a collection set point for the collection of objects having a first phenotype and the second being a set point for the collection of objects having a second phenotype.

Claims

1. An automated particle sorter, comprising:

a fluid flow path for passage of biological particles, which comprises an optical cuvette;

a fluid ring over an end of said optical cuvette, wherein said fluid ring injects fluid into a particle stream as it exits said optical cuvette;

a switch that alters the position of a collection conduit between two set points for sorting of particles according to their level of fluorescence;

a light irradiation system comprising a light source that emits light through said cuvette at a wavelength that causes fluorescence excitement;

a light detection apparatus comprising at least two detection elements for measuring emitted fluorescence; and

a data processor that receives signals from said light detection elements and according to pre-set parameters, controls said switch to physically sort said particles.

2. The particle sorter of claim 1, wherein said light irradiation system comprises a laser, a filter and focusing optics.

3. The particle sorter of claim 1, wherein said light detection apparatus comprises one or more light separation elements.

4. The particle sorter of claim 3, wherein said light separation elements are diachroic mirrors that reflect fluorescent light at a wavelength below said wavelength that causes fluorescence excitement.

5. The particle sorter of claim 4, wherein said light detection elements comprise two or more photomultiplier tubes.

6. The particle sorter of claim 5, wherein said switch is a switch that moves a collection conduit.

7. The particle sorter of claim 1, wherein said collection conduit comprises two tubes separated by a thin membrane.

8. The particle sorter of claim 1, wherein said fluid flow path is an elongated member of square or rectangular cross-sectional geometry, wherein the positions of said particles in the particles are constrained.

9. An automated particle sorter, comprising:

a fluid flow path for passage of biological particles, which comprises an optical cuvette and a high density particle chamber and a low density particle chamber, wherein the high density particle chamber and low density particle chambers are connected by a fluid valve controlled by a data processor, and particle density in said fluid flow path is maintained at a predetermined level by opening and closing of said fluid valve;

a switch that alters the position of a collection conduit between two set points for sorting of particles according to their level of fluorescence;

a light irradiation system comprising a light source that emits light through said cuvette at a wavelength that causes fluorescence excitement;

a light detection apparatus comprising at least two detection elements for measuring emitted fluorescence; and

a data processor that receives signals from said light detection elements and according to pre-set parameters, controls said switch to physically sort said particles.

10. The particle sorter of claim 9, wherein said low density particle chamber comprises a magnetic stir bar set on a pin.

11. A method of sorting biological particles according to their level of fluorescence, the method comprising:

suspending said particles in solution;

moving said suspension through a liquid flow path comprising an optical cuvette through which a light is emitted at a wavelength that excites a fluorescent compound on the particles;

detecting the level of fluorescence on said particles by at least two light detection elements, wherein a signal from said light detection elements is received by a data processor that controls a switch that alters the position of a collection conduit between two set points;

merging said suspension with a high velocity fluid flow as said suspension exits said optical cuvette that forces the particles into said collection conduit; and

moving said collection conduit between said two set points in accordance with the level of fluorescence associated with said particles.

12. The method of claim 11, wherein said particles are whole animals of from 10 to 105 cells in size.

13. The method of claim 12, wherein said whole animals are viable embryos.

14. The method of claim 12, wherein said fluorescent compound is a protein expressed by and associated with said animal.

15. The method of claim 12, wherein said animals are Drosophila embryos expressing green fluorescent protein.