Digital analyte detection system

• US 5,329,461 A
• Filed: 07/23/1992
• Issued: 07/12/1994
• Est. Priority Date: 07/23/1992
• Status: Expired due to Term

• Current Assignee: Acrogen, Inc.
• Original Assignee: Acrogen, Inc.
• Inventors: Allen, Fritz, Niemczyk, Thomas
• Primary Examiner(s): Harvey, Jack B.
• Assistant Examiner(s): CHOI, KYLE JAEHUN
• Application Number:
  US07/918,357
• Time in Patent Office: 719 Days
• Field of Search: 364/497, 364/499, 364/555, 364/502, 356/237, 324/71.4
• US Class Current: 702/26
• CPC Class Codes: G01N 15/1433 using image recognition
• 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: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/10064 Fluorescence image
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/11 Region-based segmentation
• CPC Class Codes: G06V 20/695 Preprocessing, e.g. image segmentation

Abstract

A system and method for digitally detecting the presence of analyte particles within a sample. Each analyte particle is disposed to emit an optical response upon illumination in a known manner. The digital analyte detection system includes optical apparatus for illuminating a multiplicity of distinct pixel regions within the sample so as to induce each of the analyte particles included therein to emit at least one optical response. The pixel regions are dimensioned such that the number of analyte particles included within each region is less than a maximum detection threshold proportional to variability of the optical responses. The digital detection system further includes apparatus for measuring the emitted signal emitted from each pixel region. A data processing network receives the measurements of the emitted signals and, based on the measurements, counts the number of analyte particles within each pixel region so as to determine the number of analyte particles within the sample.

Claims

1. A system for detecting the presence of a small number of analyte particles within a sample, said sample being divided into a multiplicity of distinct pixel regions where light emitted in each of said pixel regions is related to the amount of analyte in each of said pixel regions, comprising:

a plurality of pixel regions dimensioned such that the number of said analyte particles included within each of said pixel regions is less than a maximum detection threshold proportional to variation in optical responses emitted by said analyte particles;

means for measuring aggregate fluorescent energy from each of said pixel regions relative to an ambient noise threshold, wherein the aggregate fluorescent energy from each pixel region is proportional to said optical responses of analyte particles included therein; and

data processing means for receiving said measurements and, based on said measurements, counting the number of analyte particles within each of said pixel regions in order to determine the number of said analyte particles within said sample.

2. The system of claim 1 wherein said data processing means includes means for dividing each of said measurements by a value corresponding to a magnitude of one of said optical responses;

whereby the integer nearest the result of each division corresponds to the number of said analyte particles within one of said pixel regions.

3. The system of claim 1 further including means for illuminating each pixel region, wherein said measuring means includes wavelength isolation means for separating said optical responses from light provided by said illuminating means.

4. The system of claim 3 wherein said means for illuminating includes laser beam means for providing a focused illumination beam at optical wavelengths primarily confined to a first spectral range.

5. The system of claim 4 wherein said means for wavelength isolation includes an optical filter tuned to reject light included within said first spectral range.

6. The system of claim 5 wherein each of said pixel regions are of substantially equivalent area and are arranged contiguously within said sample, and wherein said illumination means further includes raster scanning means for sequentially illuminating each of said pixel regions with said focused illumination beam for a predefined time interval.

7. The system of claim 6 wherein said measuring means includes a photodetector for generating a detection signal in response to illumination by photons within each of said optical responses.

8. The system of claim 7 wherein said measuring means includes a detector circuit addressed by said detection signal for counting the number of photons emitted from each of said pixel regions to obtain pixel counts, said measuring means further including an electronic memory for storing said pixel counts.

9. The system of claim 1 wherein said data processing means includes means for generating an array of data values wherein each of said data values corresponds to said measured emitted signal from one of said pixel regions.

10. The system of claim 9 wherein said data processing means further includes:

means for calculating a background noise level based on said data values, and

means for comparing each of said data values to said background noise level in order to determine whether at least one of said analytes is included within the pixel region associated therewith.

11. The system of claim 10 wherein said data processing means further includes:

means for calculating a background noise level based on said data values,

means for arranging in descending order a set of said data values which exceed said noise level by an interest threshold, and

selecting the largest of said arranged set of data values whereby the pixel region associated with said selected data value becomes a nucleus of a first of said pixel clusters.

12. The system of claim 11 wherein said data processing means further includes:

means for identifying data values within said arranged set of data values associated with pixel regions proximate said nucleus, and

means for grouping said identified data values into said first pixel cluster.

13. The system of claim 12 wherein said data processing means further includes means for designating the largest of said arranged data values associated with a pixel region not included within said first cluster as a nucleus of a second of said pixel clusters.

14. A system for digitally detecting the presence of analyte particles within a sample in which each analyte particle is disposed to emit an optical response upon being illuminated in a predetermined manner, wherein the number of said analyte particles is less than a maximum detection threshold proportional to variability in said optical responses, comprising:

means for illuminating said sample in order to induce said analyte particles to emit said optical responses;

means for collecting aggregate fluorescent energy comprised of said optical responses; and

means for measuring said collected aggregate fluorescent energy and for comparing said measured aggregate fluorescent energy with a detection threshold in order to count the number of analyte particles within said sample.

15. The system of claim 14 wherein said means for measuring said aggregate fluorescent energy includes means for dividing said measured aggregate fluorescent energy by a value corresponding to a magnitude of said optical responses;

whereby the integer nearest said divided measurement corresponds to the number of analyte particles within said sample.

16. The system of claim 15 wherein

said means for illuminating produces illuminating light at frequencies distinct from optical frequencies associated with said optical responses, and wherein said means for collecting includes means for wavelength isolation from said illuminating light said collected optical responses.

17. The system of claim 16 wherein said means for illuminating includes laser beam means for providing a focused illumination beam at wavelengths confined to a first spectral range.

18. The system of claim 17 wherein said wavelength isolation means includes an optical filter tuned to reject light included within said first spectral range.

19. A method for detecting the presence of a small number of analyte particles within a sample, said sample being divided into a multiplicity of distinct pixel regions where light emitted in each of said pixel regions is related to the amount of analyte in each of said pixel regions, comprising the steps of:

dimensioning each of said pixel regions such that the number of said analyte particles included within each of said pixel regions is less than a maximum detection threshold proportional to variation in optical responses emitted by said analyte particles;

measuring aggregate fluorescent energy from each of said pixel regions relative to an ambient noise threshold, wherein the aggregate fluorescent energy from each pixel region is proportional to said optical responses of analyte particles included therein; and

counting, based on said measurements, the number of analyte particles within each of said pixel regions in order to determine the number of said analyte particles within said sample.

20. The method of claim 19 further including the step of dividing each of said measurements by a value corresponding to a magnitude of said optical responses;

whereby the integer nearest each of said divided measurements corresponds to the number of said analyte particles within one of said pixel regions.

21. The method of claim 19 further including the step of illuminating each of said pixel regions with illuminating light, wherein:

said step of illuminating includes the step of producing illuminating light at frequencies distinct from optical frequencies associated with said optical responses, and

said step of measuring includes the step of filtering said optical responses from said illuminating light.

22. The method of claim 21 wherein said step of illuminating further includes the step of providing a focused illumination beam at wavelengths primarily confined to a first spectral range.

23. The method of claim 19 further including the step of preparing said sample of analyte particles wherein said step of preparing includes the steps of:

binding analytes to a surface by means of a specific binding pair member;

forming analyte particles by contacting said analytes with a plurality of fluorphor labels disposed to combine with said analyte particles in a predetermined manner; and

removing said fluorphor labels not specifically bound to an analyte from the vicinity of said surface.

24. The method of claim 23 wherein said step of attaching includes the step of binding a binding member complementary to said analyte upon said surface.

25. The method of claim 24 wherein said binding member comprises an antibody specific to said analyte.

26. The method of claim 23 wherein each of said analytes is bound to a magnetic bead, and wherein said step of attaching includes the step of applying a magnetic field in the vicinity of said surface so as to induce contact between said analytes and said surface.

27. The method of claim 19 wherein said step of counting further includes the step of generating an array of data values wherein each of said data values corresponds to said measured emitted signal from one of said pixel regions.

28. The method of claim 27 wherein said step of counting further includes the steps of:

calculating a background noise level based on said data values, and

comparing each of said data values to said background noise level in order to determine whether at least one of said analyte particles is included within the pixel region associated therewith.

29. The method of claim 28 wherein said step of counting further includes the steps of:

calculating a background noise level based on said data values,

arranging in descending order a set of said data values which exceed said noise level by an interest threshold, and

selecting the largest of said arranged set of data values whereby the pixel region associated with said selected data value becomes a nucleus of a first of said pixel clusters.

30. The method of claim 29 wherein said step of counting further includes the steps of:

identifying data values within said arranged set of data values associated with pixel regions proximate said nucleus, and

grouping said identified data values into said first pixel cluster.

31. The method of claim 30 wherein said step of counting further includes the step of designating the largest of said arranged data values associated with a pixel region not included within said first cluster as a nucleus of a second of said pixel clusters.

32. A method for digitally detecting the presence of molecular analyte particles within a sample in which each analyte particle is disposed to emit an optical response upon being illuminated in a predetermined manner, wherein the concentration of said analyte particles is less than a maximum detection threshold proportional to variability in said optical responses, comprising the steps of:

illuminating said sample in order to induce said analyte particles to emit said optical responses;

collecting an aggregate fluorescent energy comprised of said optical responses; and

measuring said collected aggregate fluorescent energy and comparing said measured aggregate fluorescent energy with a detection threshold in order to count the number of analyte particles within said sample.

33. The method of claim 32 wherein said step of measuring said collected aggregate fluorescent energy includes the step of dividing said measurement by a value corresponding to one of said optical responses;

whereby said divided measurement corresponds to the number of analyte particles within said sample.

34. A method of locating and counting discrete entities within a chemical sample comprising the steps of:

storing an array of measurement values indicating presence and absence of said entities in each of a corresponding array of pixel regions of said sample;

sorting said measurement values in accordance with said indicated presence of said entities;

selecting a pixel region corresponding to a first of said measurement values as a nucleus of a first pixel cluster; and

grouping pixel regions associated with said sorted measurement values into said first pixel cluster wherein said first pixel cluster meets predefined size criteria and signifies the presence of at least one of said entities.

35. The method of claim 34 wherein said step of sorting further includes the steps of:

calculating a background noise level based on said data values; and

arranging in descending order a set of said data values which exceed said noise level by an interest threshold.

36. The method of claim 35 wherein said selected first measurement value corresponds to the largest of said arranged set of data values.

37. The method of claim 36 wherein said step of grouping further includes the steps of:

identifying data values within said arranged set of data values associated with pixel regions proximate said nucleus of said first pixel cluster, and

grouping said identified data values into said first pixel cluster.

38. The method of claim 37 wherein said step of identifying further includes the steps of:

storing a set of radii indicative of whether pixel regions associated with said arranged data values exist in predefined locations proximate said first nucleus;

calculating, based on said stored radii, an optimal cluster area circumscribing said first nucleus wherein said identified data values correspond to those arranged data values associated with pixel regions within said optimal cluster area.

39. The method of claim 38 wherein said step of grouping further includes the step of designating the largest of said arranged data values associated with a pixel region not included within said first cluster as a nucleus of a second pixel cluster.