COMPACT ANALYZER WITH SPATIAL MODULATION AND MULTIPLE INTENSITY MODULATED EXCITATION SOURCES

• US 20130037726A1
• Filed: 08/09/2011
• Published: 02/14/2013
• Est. Priority Date: 08/09/2011
• Status: Active Grant

• Current Assignee: Xerox Corporation (Xerox Holdings Corp.)
• Original Assignee: Palo Alto Research Center, Inc. (Xerox Holdings Corp.)
• Inventors: Kiesel, Peter, Martini, Joerg
• Granted Patent: US 9,029,800 B2
• Time in Patent Office: Days
• US Class Current: 250/458.1
• CPC Class Codes: G01J 2003/104 Monochromatic plural sources
• CPC Class Codes: G01J 3/36 Investigating two or more bands of a spectrum by separate detectors
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/145 by pattern of light, e.g. fringe pattern
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/65 Raman scattering
• CPC Class Codes: G01N 2201/0691 Modulated (not pulsed supply)

Abstract

A compact analyzer includes a flow cell having a flow channel through which a sample is made to pass. First and second light sources are arranged to emit first and second excitation light into first and second overlapping portions of the flow channel, respectively. The first excitation light stimulates a first light emission from particles of a first particle type that may be present in the sample; the second excitation light stimulates a second light emission from particles of a second particle type. A detector receives the first and second light emission from the corresponding particles present in the sample in a detection portion of the flow channel, and provides a detector output based on the received light emission. The light sources are modulated at different frequencies so that a frequency analysis of the detector output can provide separate information about the first and second particle types.

Claims

1. An apparatus for analyzing a sample that may comprise particles of at least a first and second particle type, the apparatus comprising:

a flow channel through which the sample can pass;

a first light source adapted to emit first excitation light into a first excitation portion of the flow channel, the first excitation light adapted to stimulate a first light emission from particles of the first particle type;

a second light source adapted to emit second excitation light into a second excitation portion of the flow channel, the second excitation light adapted to stimulate a second light emission from particles of the second particle type; and

a detector disposed to receive at least a portion of the first and second light emission from the corresponding particles that may be present in the sample in a detection portion of the flow channel, the detection portion overlapping with the first and second excitation portions of the flow channel, the detector adapted to provide a detector output based on the received light emission;

wherein the first light source is adapted to emit the first excitation light at a first modulation frequency ν

1, and the second light source is adapted to emit the second excitation light at a second modulation frequency ν

2 different from ν

1.

2. The apparatus of claim 1, further comprising:

a spatial filter having a pattern of variable transmission, the spatial filter being disposed between the detection portion of the flow channel and the detector, the spatial filter further being adapted to transmit light emanating from the particles that may be present in the flow channel by varying amounts as such particles travel along the detection portion of the flow channel.

3. The apparatus of claim 2, wherein the first and second modulation frequencies are each faster than a modulation associated with the variable transmission of the spatial filter.

4. The apparatus of claim 2, wherein the first and second excitation portions of the flow channel substantially overlap with each other and with the detection portion of the flow channel, and wherein the spatial filter spans the detection portion.

5. The apparatus of claim 1, further comprising:

a controller coupled to the first and second light sources, the controller adapted to modulate the first and second light sources at the modulation frequencies ν

1, ν

2 respectively.

6. The apparatus of claim 1, wherein the first excitation light has a first peak wavelength, and the second excitation light has a second peak wavelength different from the first peak wavelength.

7. The apparatus of claim 1, wherein the first excitation light is adapted to stimulate little or no light emission from particles of the second type, and the second excitation light is adapted to stimulate little or no light emission from particles of the first type.

8. The apparatus of claim 1, wherein the first light emission comprises fluorescence emitted by particles of the first particle type when exposed to the first excitation light, the apparatus further comprising:

an optical filter disposed between the detector and the flow channel, the optical filter adapted to preferentially transmit the first light emission and to preferentially block the first excitation light.

9. The apparatus of claim 8, wherein the second light emission comprises fluorescence emitted by particles of the second particle type when exposed to the second excitation light, and wherein the optical filter is also adapted to preferentially transmit the second light emission and to preferentially block the second excitation light.

10. The apparatus of claim 1, further comprising:

a signal processing unit coupled to the detector to receive the detector output, wherein the signal processing unit provides a system output based on the detector output, the system output providing a first measure of the particles of the first particle type in the sample and a second measure of the particles of the second particle type in the sample.

11. An apparatus for analyzing a sample that may comprise particles of at least a first and second particle type, the apparatus comprising:

a flow channel through which the sample can pass;

a first light source adapted to emit first excitation light into a first excitation portion of the flow channel, the first excitation light adapted to stimulate a first light emission from particles of the first particle type and to stimulate little or no light emission from particles of the second type;

a second light source adapted to emit second excitation light into a second excitation portion of the flow channel, the second excitation light adapted to stimulate a second light emission from particles of the second particle type and to stimulate little or no light emission from particles of the first type;

a detector disposed to receive the first and second light emission from the corresponding particles that may be present in the sample in a detection portion of the flow channel, the detection portion overlapping with the first and second excitation portions of the flow channel, the detector adapted to provide a detector output based on the received light emission;

an optical filter disposed between the detector and the flow channel, the optical filter adapted to preferentially transmit the first and second light emission and to preferentially block the first and second excitation light;

a spatial filter having a pattern of variable transmission, the spatial filter being disposed between the detection portion of the flow channel and the detector, the spatial filter further being adapted to transmit light emanating from the particles that may be present in the flow channel by varying amounts as such particles travel along the detection portion of the flow channel;

a controller coupled to the first and second light sources, the controller adapted to modulate the first light source at a first modulation frequency ν

1, the controller also adapted to modulate the second light source at a second modulation frequency ν

2 different from ν

1; and

a signal processing unit coupled to the detector to receive the detector output, wherein the signal processing unit provides a system output based on the detector output, the system output providing a first measure of the particles of the first particle type in the sample and a second measure of the particles of the second particle type in the sample.

12. A method for analyzing a sample that may comprise particles of at least a first and second particle type, the method comprising:

passing the sample through a flow channel;

illuminating the sample in a first excitation portion of the flow channel with first excitation light from a first light source while the sample flows through the flow channel, the first excitation light adapted to stimulate a first light emission from particles of the first particle type;

illuminating the sample in a second excitation portion of the flow channel with second excitation light from a second light source while the sample flows through the flow channel, the second excitation light adapted to stimulate a second light emission from particles of the second particle type; and

detecting at least a portion of the first and second light emission with a detector adapted to provide a detector output based on the received light emission;

wherein the illuminating is carried out such that the first excitation light is modulated at a first modulation frequency ν

1, and the second excitation light is modulated at a second modulation frequency ν

2 different from ν

1.

13. The method of claim 12, wherein the first and second excitation portions of the flow channel overlap with each other and with a detection portion of the flow channel, the method further comprising:

spatially modulating light emitted from particles in the detection portion of the flow channel, such that the first and second light emission is detected by the detector with a third modulation frequency different from the first and second modulation frequencies, the third modulation frequency being based on a flow rate of the sample through the flow channel.

14. The method of claim 13, wherein the spatially modulating includes providing a mask between the detection portion of the flow channel and the detector.

15. The method of claim 14, wherein the spatially modulating further includes imaging the mask onto the detection portion of the flow channel.

16. The method of claim 12, wherein the modulation of the first excitation light is periodic, and the modulation of the second excitation light is periodic.

17. The method of claim 13, wherein the modulation of the first excitation light is periodic, the modulation of the second excitation light is periodic, and the spatial modulation of the light emitted from particles in the detection portion of the flow channel is periodic.

18. The method of claim 12, wherein the first light emission has a first intrinsic response time and an associated first intrinsic frequency, and wherein the first modulation frequency is less than the first intrinsic frequency.

19. The method of claim 12, wherein the second light emission has a second intrinsic response time and an associated second intrinsic frequency, and wherein the second modulation frequency is less than the second intrinsic frequency.

20. The method of claim 12, further comprising:

performing a frequency analysis of the detector output, the frequency analysis including identifying one or more peaks in a frequency spectrum of the detector output.

21. A method, comprising:

using n types of absorption-encoded micro beads, wherein each type of absorption-encoded micro bead including amounts of k dyes in a proportional relationship that is different from proportional relationships of the k dyes of other types of absorption-encoded micro beads, each of the k dyes having an excitation spectrum different from excitation spectra of others of the k dyes;

stimulating light emission from each of the k dyes present in a sample suspected of containing at least one type of the absorption encoded micro beads, the stimulating including illuminating the sample with k types of light, each type of light having a spectrum that overlaps an excitation spectrum of one of the k dyes to a greater extent than excitation spectra of others of the k dyes;

detecting at least a portion of the light emission; and

identifying each type of absorption-encoded micro bead present in the sample based on the light emission.

22. The method of claim 21, wherein:

the k dyes comprise a first dye and a second dye, the first dye having a first excitation spectrum and the second dye having a second excitation spectrum;

stimulating the light emission comprising stimulating a first light emission from the first dye and stimulating a second light emission from the second dye; and

detecting the light emission comprises detecting at least a portion of the first light emission and detecting at least a portion of the second light emission.

23. The method of claim 22, wherein:

a proportional relationship of the first and second dyes of a first type of absorption-encoded micro bead comprises 100% of the first dye and 0% of the second dye;

a proportional relationship of the first and second dyes of a second type of absorption-encoded micro bead comprises 0% of the first dye and 100% of the second dye; and

a proportional relationship of the first and second dyes of a third type of absorption-encoded micro bead comprises 50% of the first dye and 50% of the second dye.

24. The method of claim 21, wherein a first dye of the k dyes comprises PerCP and a second dye of the k dyes comprises APC.

25. The method of claim 21, wherein a first dye of the k dyes comprises Cy3 and a second dye of the k dyes comprises Cy5.

26. The method of claim 21, wherein stimulating the light emission comprises illuminating the sample with k types of excitation light, each type of excitation light adapted to stimulate light emission more predominantly from one of the k dyes, respectively, and less predominantly from others of the k dyes.

27. The method of claim 26, wherein illuminating the sample with k types of excitation light comprises illuminating the sample with k types of excitation light, the k types of excitation light modulated using k modulation frequencies, respectively.

28. A method of analyzing a sample, comprising:

illuminating the sample, the sample including at least one of n types of absorption-encoded micro beads, each type of absorption-encoded micro bead comprising amounts of k dyes in a proportional relationship that is different from proportional relationships of the k dyes of other types of absorption-encoded micro beads, each of the k dyes having an excitation spectrum different from excitation spectra of others of the k dyes, the illuminating comprising illuminating the sample with k types of excitation light, each type of excitation light adapted to stimulate light emission from one of the k dyes, respectively;

detecting at least a portion of the light emission; and

identifying each type of absorption-encoded micro bead present in the sample based on the light emission.

29. The method of claim 28, wherein:

k equals 2; and

n is greater than 2.

30. The method of claim 28, wherein:

the k dyes comprise a first dye and a second dye, the first dye having a first excitation spectrum and the second dye having a second excitation spectrum;

illuminating the sample comprises illuminating the sample using a first type of excitation light adapted to stimulate first light emission from the first dye and illuminating the sample using a second type of excitation light adapted to stimulate second light emission from the second dye; and

detecting at least a portion of the light emission comprises detecting at least a portion of the first light emission and detecting at least a portion of the second light emission.

31. The method of claim 30, wherein:

a proportional relationship of the first and second dyes of a first type of absorption-encoded micro bead comprises 100% of the first dye and 0% of the second dye;

a proportional relationship of the first and second dyes of a second type of absorption-encoded micro bead comprises 0% of the first dye and 100% of the second dye; and

a proportional relationship of the first and second dyes of a third type of absorption-encoded micro bead comprises 50% of the first dye and 50% of the second dye.

32. The method of claim 28, wherein a first dye of the k dyes comprises PerCP and a second dye of the k dyes comprises APC.

33. The method of claim 28, wherein a first type of absorption-encoded micro bead has first set of physical characteristics and a second type of absorption-encoded micro bead has a second set of physical characteristics.

34. The method of claim 28, wherein a first type of absorption-encoded micro bead has a first size and a second type of absorption-encoded micro bead has a second size different from the first size.

35. The method of claim 28, wherein a spectrum of the first light emission substantially overlaps a spectrum of the second light emission.

36. A method of fabricating micro beads, comprising encoding n types of micro beads to form absorption-encoded micro beads, wherein each type of absorption-encoded micro bead has an excitation spectrum distinguishable from spectra of other types of absorption-encoded absorption-encoded micro beads, each type of absorption-encoded micro bead including amounts of the k dyes in a proportional relationship that is different from proportional relationships of the k dyes of other types of absorption-encoded micro beads, each of the k dyes having an excitation spectrum different from excitation spectra of others of the k dyes.

37. The method of claim 36, wherein:

a proportional relationship of the first and second dyes of a first type of absorption-encoded micro bead comprises 100% of the first dye and 0% of the second dye;

a proportional relationship of the first and second dyes of a second type of absorption-encoded micro bead comprises 0% of the first dye and 100% of the second dye; and

a proportional relationship of the first and second dyes of a third type of absorption-encoded micro bead comprises 50% of the first dye and 50% of the second dye.

38. The method of claim 36, wherein:

k equals 2; and

n is greater than 2.