US5329461A — Digital analyte detection system

Claim 1

Independent Claim 165 words

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.

Claim 2

Independent Claim 165 words

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.

Claim 3

Dependent Claim 54 words

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.

Claim 4

Dependent Claim 33 words

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.

Claim 5

Dependent Claim 30 words

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.

Claim 6

Dependent Claim 25 words

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.

Claim 7

Dependent Claim 51 words

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.

Claim 8

Dependent Claim 29 words

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.

Claim 9

Dependent Claim 48 words

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.

Claim 10

Dependent Claim 37 words

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.

Claim 11

Dependent Claim 57 words

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.

Claim 12

Dependent Claim 76 words

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.

Claim 13

Dependent Claim 44 words

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.

Claim 14

Dependent Claim 43 words

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.

Claim 15

Independent Claim 115 words

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.

Claim 16

Independent Claim 115 words

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.

Claim 17

Dependent Claim 51 words

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.

Claim 18

Dependent Claim 43 words

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.

Claim 19

Dependent Claim 28 words

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.

Claim 20

Dependent Claim 24 words

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

Claim 21

Independent Claim 158 words

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.

Claim 22

Independent Claim 158 words

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.

Claim 23

Dependent Claim 49 words

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.

Claim 24

Dependent Claim 59 words

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.

Claim 25

Dependent Claim 29 words

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.

Claim 26

Dependent Claim 79 words

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.

Claim 27

Dependent Claim 25 words

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.

Claim 28

Dependent Claim 16 words

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

Claim 29

Dependent Claim 47 words

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.

Claim 30

Dependent Claim 39 words

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.

Claim 31

Dependent Claim 57 words

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.

Claim 32

Dependent Claim 75 words

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.

Claim 33

Dependent Claim 43 words

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.

Claim 34

Dependent Claim 44 words

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.

Claim 35

Independent Claim 113 words

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.

Claim 36

Independent Claim 113 words

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.

Claim 37

Dependent Claim 46 words

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.

Claim 38

Independent Claim 109 words

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.

Claim 39

Independent Claim 109 words

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.

Claim 40

Dependent Claim 45 words

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.

Claim 41

Dependent Claim 22 words

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

Claim 42

Dependent Claim 48 words

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.

Claim 43

Dependent Claim 73 words

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.

Claim 44

Dependent Claim 42 words

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.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1997-07-11	1998-01-10	1998-07-12	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2001-07-11	2002-01-10	2002-07-12	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2005-07-11	2006-01-10	2006-07-12	$7700.00	Window Closed / Lapsed

Digital analyte detection system

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