Method and apparatus for reading reporter labeled beads

• US 20060029267A1
• Filed: 03/11/2005
• Published: 02/09/2006
• Est. Priority Date: 08/25/2000
• Status: Active Grant

• Current Assignee: Cytek Biosciences Inc
• Original Assignee: Amnis Corporation (DiaSorin S.p.A.)
• Inventors: Basiji, David A., Perry, David J., Ortyn, William E., Bauer, Richard A., Frost, Keith L., Finch, Rosalynde J.
• Granted Patent: US 7,567,695 B2
• Time in Patent Office: Days
• US Class Current: 382/129
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 60/10 for identifying library members
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1475 using image analysis for extracting features of the particle
• CPC Class Codes: G01N 2015/1447 Spatial selection
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 2015/1479 Using diffuse illumination or excitation
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G02B 27/0012 Optical design, e.g. procedures, algorithms, optimisation routines
• CPC Class Codes: G02B 27/1013 for colour or multispectral image sensors, e.g. splitting an image into monochromatic image components on respective sensors spectral imaging systems G01J
• CPC Class Codes: G02B 27/126 The splitting element being a prism or prismatic array, including systems based on total internal reflection
• CPC Class Codes: G02B 27/144 using partially transparent surfaces without spectral selectivity G02B27/147 takes precedence
• CPC Class Codes: G02B 27/145 having sequential partially reflecting surfaces
• CPC Class Codes: G02B 27/148 : including stacked surfaces having at least one double-pass partially reflecting surface
• CPC Class Codes: G02B 7/28 : Systems for automatic generation of focusing signals

Abstract

Combinatorially-synthesized deoxyribonucleic acid (DNA) oligonucleotides attached to encoded beads that are hybridized to amplified and labeled genomic DNA or ribonucleic acid (RNA) are analyzed using a flow imaging system. Oligonucleotides and corresponding reporters are bound to the surfaces of a plurality of small beads such that different beads bear different oligo sequences. Each bead bears a unique optical signature comprising a predefined number of unique reporters, where each reporter comprises a predefined combination of different fluorochromes. The composite spectral signature in turn identifies the unique nucleotide sequence of its attached oligo chains. This optical signature is rapidly decoded using an imaging system to discriminate the different reporters attached to each bead in a flow in regard to color and spatial position on the bead.

Claims

1. A method for imaging and decoding a plurality of encoded beads comprising the steps of:

(a) collecting light from an encoded bead along a collection path;

(b) dispersing the light that is traveling along the collection path into a plurality of light beams, as a function of a plurality of different discriminable characteristics of the light;

(c) focussing each of the plurality of light beams to produce a respective image corresponding to that light beam, thereby generating a plurality of images, each image being indicative of an identity of said encoded bead;

(d) detecting the plurality of images;

(e) analyzing the plurality of images to determine the identity of said encoded bead; and

(f) repeating steps (a)-(e) for successive encoded beads from the plurality of encoded beads.

2. The method of claim 1, wherein the step of dispersing comprises the steps of dividing the light into the plurality of light beams as a function of a wavelength.

3. The method of claim 1, wherein the step of analyzing the plurality of images comprises the steps of generating a plurality of signals, each signal thus generated indicating the presence of a different discriminable characteristic of the light.

4. The method of claim 1, wherein the step of analyzing the plurality of images comprises the step of constructing a sequence library based on each encoded bead that is decoded.

5. The method of claim 1, further comprising the step of illuminating the plurality of encoded beads with light while the encoded beads are moving.

6. The method of claim 1, wherein the step of analyzing the plurality of images comprises the steps of:

(a) determining dispositions of reporters associated with the bead;

(b) determining a signature of each reporter associated with the bead based upon the dispositions of the reporters in the plurality of images collected from each encoded bead; and

(c) decoding each bead as a function of each signature associated with the bead.

7. The method of claim 1, further comprising the step of providing redundant reporters for each encoded bead.

8. The method of claim 1, further comprising the step of disregarding the identity of an encoded bead if the plurality of images for that encoded bead indicate that fewer than a predetermined number of reporters are associated with the encoded bead.

9. The method of claim 1, wherein the step of analyzing comprises the step of referring to an encoded bead legend that identifies each encoded bead as a function of optically discriminable reporters associated with each encoded bead.

10. The method of claim 1, further comprising the step of disregarding the identity of each encoded bead if the analysis of the plurality of images determined that an encoded bead has not experienced a binding event.

11. The method of claim 1, wherein the step of analyzing comprises the step of de-convolving the images if the step of dispersing convolves the plurality of light beams.

12. The method of claim 1, wherein the step of dispersing comprises the step of providing an image corresponding to a binding signal produced by the encoded bead.

13. A method for simultaneously imaging a plurality of reporters disposed on substantially different portions of an encoded bead to identify each unique reporter included on the encoded bead, said method comprising the steps of:

(a) receiving light from the encoded bead along a plurality of collection paths that are substantially spaced apart, such that light from the reporters disposed on the different portions of the encoded bead affect the light received therefrom; and

(b) processing the light received from the encoded bead along the plurality of collections paths to identify each unique reporter included on the encoded bead.

14. The method of claim 13, further comprising the step of providing redundant copies of each unique reporter on the encoded bead.

15. The method of claim 13, further comprising the steps of reducing the number of unique reporters necessary to encode a bead;

and selecting a library to encode a bead characterized by a length between 9-mer and 16-mer.

16. An imaging system for imaging and decoding a plurality of encoded beads to which is attached one or more compounds, each compound being associated with a unique reporter set, each reporter set including at least one reporter, comprising:

(a) a collection lens disposed so that light traveling from each encoded bead passes through the collection lens and is focussed along a collection path;

(b) a dispersing component that receives the light from the collection lens and disperses the light into a plurality of light beams, as a function of a plurality of different discriminable characteristics of the light, said plurality of different discriminable characteristics being indicative of the reporter sets associated with the encoded beads;

(c) at least one pixilated detector;

(d) an imaging lens that focuses each of the plurality of light beams on said at least one pixilated detector, producing a respective image corresponding to each of the plurality of light beams, said at least one pixilated detector providing an output signal for each respective image, each output signal indicating the reporter set associated with the encoded bead; and

(e) a signal processor coupled to receive the output signals from said at least one pixilated detector, said signal processor processing the output signals to decode each reporter set associated with the encoded bead, thereby identifying each compound attached to the encoded bead.

17. The imaging system of claim 16, wherein said signal processor is adapted to generated sequence contigs from a plurality of decoded beads.

18. The imaging system of claim 17, wherein said sequence contigs identify at least one of a genomic DNA sequence, a polymorphic allele, and an expressed gene.

19. The imaging system of claim 16, wherein said signal processor is adapted to analyze said output signals to:

(a) determine dispositions of the reporters on the encoded bead;

(b) determine a signature of each reporter associated with the encoded bead based upon the dispositions of the reporters on the bead; and

(c) determine a reporter set associated with the encoded bead based upon the reporter signatures; and

(d) identify the each compound associated with the reporter set.

20. The imaging system of claim 16, wherein said signal processor is adapted to disregard all output signals relating to a reporter if signals from an identical reporter have already been analyzed, and to disregard all output signals for an encoded bead if said signals indicate that fewer than a predetermined number of reporters are associated with the encoded bead.

21. The imaging system of claim 16, wherein said signal processor is adapted to employ an encoded bead legend that relates each unique reporter set to a specific compound.

22. The imaging system of claim 21, wherein said signal processor is adapted to disregard all output signals for an encoded bead if it is determined that the encoded bead does not correspond to said encoded bead legend.

23. The imaging system of claim 16, wherein said signal processor is adapted to de-convolve the output signals if said dispersing component convolves the plurality of light beams.

24. The imaging system of claim 16, wherein said dispersing component provides one respective image corresponding to a binding signal produced by the encoded bead.

25. The imaging system of claim 24, wherein said signal processor is adapted to disregard all output signals for an encoded bead if said one respective image indicates that an encoded bead has not experienced a binding event.

26. The imaging system of claim 16, wherein said dispersing component comprises one of a dichroic filters and a prism.

27. The imaging system of claim 16, wherein said at least one pixilated detector comprises a time delay integration (TDI) detector.

28. The imaging system of claim 16, wherein said imaging lens focuses each one of said plurality of light beams onto a different region of said at least one pixilated detector.