Detection, resolution, and identification of arrayed elements Number:7,522,762 from the United States Patent and Trademark Office (PTO) owispatent

Title: Detection, resolution, and identification of arrayed elements

Abstract: An image analysis workstation for analyzing optical thin film arrays is disclosed. One disclosed embodiment relates to individual arrays that comprise a single optical thin film test surface that provides a plurality of discretely addressable locations, each comprising an immobilized capture reagent for an analyte of interest. These are referred to herein as "arrayed optical thin film test surfaces." Preferably, an individual arrayed optical thin film test surface comprises at least 4, more preferably at least 16, even more preferably at least 32, still more preferably at least 64, and most preferably 128 or more discretely addressable locations. One or more of the discretely addressable locations may provide control signals (e.g., for normalizing signals and/or that act as positive and/or negative controls) or fiducial signals (i.e., information that is used to determine the relative alignment of the arrayed optical thin film test surface within the device.

Patent Number: 7,522,762 Issued on 04/21/2009 to Rea,   et al.

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Inventors:	Rea; Larry (Lafayette, CO), Clark; David D. (Longmont, CO), Jenison; Rob (Boulder, CO), Maul; Diana (Thomton, CO)
Assignee:	Inverness Medical-Biostar, Inc. (Waltham, MA)
Appl. No.:	10/417,883
Filed:	April 16, 2003

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Current U.S. Class:	382/141 ; 438/16
Current International Class:	G06K 9/00 (20060101); G01R 31/26 (20060101)

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References Cited [Referenced By]

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U.S. Patent Documents

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5639671	June 1997	Bogart et al.
5782361	July 1998	Kakizaki et al.
5955377	September 1999	Maul et al.
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6060237	May 2000	Nygren et al.
6287783	September 2001	Maynard et al.
6292586	September 2001	Kawakami et al.
6355429	March 2002	Nygren et al.
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6410252	June 2002	Lehmann et al.
6441894	August 2002	Manian et al.
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2003/0096302	May 2003	Yguerabide et al.
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2005/0040907	February 2005	Nebrigic

Foreign Patent Documents

	WO 01/92870		Dec., 2001		WO

Other References

Notification Of Transmittal of International Search Report and International Preliminary Examination Report dated May 1, 2006 (9 pages). cited by other . Ando, "Consistent Gradient Operators." IEEE Transactions on Pattern Analysis and Machine Intelligence, 22(3): 252-265, 2000. cited by other.

Primary Examiner: Kim; Charles
Attorney, Agent or Firm: Swanson & Bratschun L.L.C.

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Claims

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We claim:

1. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analyte; wherein said signal is determined simultaneously from each of said plurality of discrete test locations on one of said discrete arrayed optical thin film test surfaces by capturing an image of a discrete arrayed optical thin film test surface, and performing image analysis on said image to obtain a signal from each of said plurality of discrete test locations; and wherein said image analysis comprises: identifying an initial array location in the image of the arrayed optical thin film test surface, identifying whether skew or flatness is present and correcting for skew and flatness if present; thresholding the image; identifying individual test locations in the thresholded image and refining the array location by comparing an offset of the location of each test location to a predicted test location obtained from the initial array location; and obtaining a signal from each test location in the refined array.

2. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analytes; wherein the location of the arrayed optical thin film test surface on the test surface carrier is identified by a method comprising: a) acquiring an electronic image of said test surface; b) squaring said image to orient columns and rows of pixels in a substantially vertical and horizontal configuration, respectively, said squaring comprising: detecting edges of said image, said detecting comprising forming a first vector of pixel sums for a set of columns of pixels in said image; forming a second vector of pixel sums for a set of rows of pixels in said image; generating a squared first derivative of said first and second vectors; and detecting peaks in said squared first derivatives; and rotating said image; c) locating a grid of said columns and rows of said image; and d) binarizing said image using a threshold value of signal strength to identify location of spots on said grid.

3. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analytes; wherein the location of an arrayed optical thin film test surface on a test surface carrier is identified by a method comprising: a) acquiring an electronic image of said test surface; b) squaring said image to orient columns and rows of pixels in a substantially vertical and horizontal configuration, respectively; c) locating a grid of said columns and rows of said image, wherein said locating comprises detecting a grid of signal peaks corresponding to rows and columns of said grid, said detecting the grid comprising the steps of generating second derivatives of inverted pixel sums, and locating minima of said second derivatives; and d) binarizing said image using a threshold value of signal strength to identify location of spots on said grid.

4. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analytes; wherein the location of the arrayed optical thin film test surface on the test surface carrier is identified by a method comprising: a) generating gradients for an image in two substantially orthogonal directions; b) generating a squared gradient magnitude corresponding to said generated gradients; c) binarizing said gradient magnitude using a threshold value of said squared gradient magnitude; d) generating a transform image based on said gradients; e) binarizing said transform image using a threshold value of a maximum vote count for the transform of step d) to produce a thresholded transform image; f) detecting contiguous groups of pixels in said thresholded transform image; g) building a grid to correspond to a layout of said array; h) measuring signal strength at spot locations of said grid; and i) generating a table of spot positions and signals, said spot positions being indicative of a spot center.

5. The method according to claim 4, further comprising: detecting edges of said test surface prior to step a); and cropping said image to said detected edges.

6. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analytes; wherein the location of the arrayed optical thin film test surface on the test surface carrier is identified by a method comprising: a) acquiring an electronic image of said test surface; b) squaring said image to orient columns and rows of pixels in a substantially vertical and horizontal configuration, respectively; c) locating a grid of said columns and rows of said image, wherein said location is identified by using a program product comprising machine readable program code for causing a machine to perform following method steps: i) squaring said test surface to orient columns and rows of pixels in a substantially vertical and horizontal configuration, respectively, said squaring comprising using program code for causing a machine to perform the following step: detecting edges of said image wherein said detecting edges comprises using a program code for causing a machine to perform the steps of: forming a first vector of pixel sums for a set of columns of pixels in said image; forming a second vector of pixel sums for a set of rows of pixels in said image; generating a squared first derivative of said first and second vectors; and detecting peaks in said squared first derivatives; and rotating said image; ii) locating a grid of said columns and rows of said image; and d) binarizing said image using a threshold value of signal strength to identify location of spots on said grid.

7. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analytes; wherein the location of the arrayed optical thin film test surface on the test surface carrier is identified by a method comprising: a) acquiring an electronic image of said test surface; b) squaring said image to orient columns and rows of pixels in a substantially vertical and horizontal configuration, respectively; c) locating a grid of said columns and rows of said image, wherein said location is identified by using a program product comprising machine readable program code for causing a machine to perform following method steps: i) squaring said test surface to orient columns and rows of pixels in a substantially vertical and horizontal configuration, respectively; ii) locating a grid of said columns and rows of said image, said locating a grid comprising using program code for causing a machine to perform the step of detecting of a grid of signal peaks corresponding to rows and columns of said grid, said detecting of a grid of signal peaks comprising using program code for causing the machine to perform the steps of generating second derivatives of inverted pixel sums, and locating minima of said second derivatives; and d) binarizing said image using a threshold value of signal strength to identify location of spots on said grid.

8. A method for determining the presence or amount of a plurality of analytes in one or more samples using a test surface carrier comprising a plurality of discrete arrayed optical thin film test surfaces, each arrayed optical thin film test surface comprising a plurality of discrete test locations comprising a capture reagent to immobilize for detection of one of said analytes, said method comprising: contacting each of said discrete arrayed optical thin film test surfaces with a sample to be tested for said plurality of analytes, whereby said analytes, if present, are immobilized at a corresponding test location; removing unbound sample components from each of said discrete arrayed optical thin film test surfaces; and determining a signal from each of said plurality of discrete test locations on each of said discrete arrayed optical thin film test surfaces, wherein each of said signals is related to a change in mass or optical thickness of the corresponding optical thin film, each of said signals comprising light reflected from the surface that has undergone a change in the polarization state, phase or interference color, and relating each of the signals to the presence or amount of one of said plurality of analytes; wherein the location of the arrayed optical thin film test surface on the test surface carrier is identified by using a program product comprising a machine readable program code for causing a machine to perform the following method steps: a) generating gradients for an image in two substantially orthogonal directions; b) generating a squared gradient magnitude corresponding to said generated gradients; c) binarizing said gradient magnitude using a threshold value of said squared gradient magnitude; d) generating a transform image based on said gradients; e) binarizing said transform image using a threshold value of a maximum vote count for the transform of step d) to produce a thresholded transform image; f) detecting contiguous groups of pixels in said thresholded transform image; g) building a grid to correspond to a layout of said array; h) measuring signal strength at spot locations of said grid; and i) generating a table of spot positions and signals, said spot positions being indicative of a spot center.

9. The method according to claim 8, wherein said program code causes a machine to further perform the following method steps: detecting edges of said test surface prior to step a); and cropping said image to said detected edges.

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Description

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FIELD OF INVENTION

This invention relates to the optical detection, resolution, and identification of an array of elements, preferably for use on an optical thin film surface

BACKGROUND OF INVENTION

The following description of the background of the invention is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.

In the semiconductor field, films formed by vacuum evaporation, vapour deposition, spin coating or dip coating are commonly used at various stages of the semiconductor fabrication process. Control and monitoring of the actual thickness and physical properties of thin film layers is absolutely essential to the function of the devices created using this technology. These characteristics must often be monitored during and after fabrication. "Optical thin film determination" refers to methods for determining the thicknesss of one or multiple thin layer(s) formed on a substrate surface. Such "thin films" range from about 1 nm to about 100 .mu.m in thickness.

Typically, optical thin film measurements rely on changes in one or more characteristics of light reflected from a substrate comprising an "optical thin film test surface." By this is meant that the surface is reflective of incident light, and is configured and arranged by selection of refractive index (n) and absorption coefficient (k) for generation of a signal directly due to a change in mass or thickness upon the surface. The signal is obtained by illuminating the surface with light; light is reflected from the surface or transmitted through the surface, and any thin film upon the surface will alter the color, ellipticity, and/or intensity of one or more wavelengths in the reflected or transmitted light due to an interference effect. This extent of the alteration, and hence the signal obtained, depends on the mass or thickness of any surface film(s).

Devices for optical measurement of thin films generally fall into two instrument classes: reflectometers and ellipsometers. Reflectometry is based upon measurement of changes in intensity and/or color of light reflected from the optical thin film test surface; ellipsometry is based on measurement of changes of the polarisation of light reflected from the optical thin film test surface. Such methods are well known in the art. See, e.g., Tompkins and McGahan, Spectroscopic Ellipsometry and Reflectometry: A User's Guide, John Wiley and Sons, 1999, which discusses the nature of optical constants of materials, instrumental aspects of reflectometers, ellipsometric spectra, and single-wavelength ellipsometry, as well as analytical approaches for collecting and analyzing ellipsometric and reflectance data.

Because of the ability of such methods and devices to sensitively detect changes in film thickness at molecular dimensions, the application of optical thin film measurements to biological systems has become well established. For example, devices and methods for direct detection of binding reactions (e.g., in immunoassay, nucleic acid hybridization, etc.) has been described. See, e.g., U.S. Pat. Nos. 6,483,585; 6,355,429; 6,287,783; 6,060,237; 5,955,377; 5,639,671; 5,631,171; 5,629,214; 5,552,272; 5,550,063; 5,494,829. While such methods do not depend upon the presence of a signal development element (e.g., a fluorometric, luminescent, or calorimetric moiety) for production of a signal, amplification methods (e.g., the catalytic production of a precipitate or the binding of particles such as latex, gold, etc.) to provide additional mass or optical thickness may be employed to enhance detection of the binding reaction.

SUMMARY OF THE INVENTION

The present invention relates to devices, compositions, and methods for manufacture and use of high-throughput thin film optical assay devices. The following sections describe hardware and software requirements for the analysis of optical thin film test surface arrays for use in medical or research applications such as genomics, proteomics, allergy panels, drug discovery, high throughput screening, pharmacogenomics, toxicogenomics, ADME screening, infectious disease panels, SNP (single nucleotide polymorphisms) analysis for a specific disease or condition, etc.

In a first aspect, the present invention relates to individual arrays that comprise a single optical thin film test surface that provides a plurality of discretely addressable locations, each comprising an immobilized capture reagent for an analyte of interest. These are referred to herein as "arrayed optical thin film test surfaces." Preferably, an individual arrayed optical thin film test surface comprises at least 4, more preferably at least 16, even more preferably at least 32, still more preferably at least 64, and most preferably 128 or more discretely addressable locations. One or more of the discretely addressable locations may provide control signals (e.g., for normalizing signals and/or that act as positive and/or negative controls) or fiducial signals (i.e., information that is used to determine the relative alignment of the arrayed optical thin film test surface within the device.

In a related aspect, the arrayed optical thin film test surfaces are contained in a larger "test surface carrier" that provides an "array of arrays" within a single housing, thereby further increasing the throughput Preferably, an individual test surface carrier comprises at least 1, more preferably at least 2, still more preferably at least 5, even more preferably at least 10, still more preferably at least 20, even more preferably at least 50, and most preferably at least 90 or more discrete arrayed optical thin film test surfaces within a single housing. Like the arrays, a test surface carrier may also be provided with fiducial locations that provide information that is used to determine the relative alignment of the optical thin film test surfaces within the test surface carrier.

The term "discrete" as used herein with regard to individual arrays refers to two or more arrays having discontinuous surfaces. The term "discretely addressable" as used herein with regard to individual locations on a single array refers to discrete areas of a surface from which a specific signal may be obtained.

Signal from discretely addressable locations on the arrayed optical thin film test surface is generated by a change in film thickness or mass as a result of a specific reaction of a target molecule with its corresponding capture reagent at a position within the array. As the film thickness or mass changes, light reflected from the surface undergoes a change in the polarization state, phase, or in interference color. Capture reagents may be small molecules, polypeptides, proteins, cyclic polypeptides, peptidomimetics, aptamers, antibodies, scFvs, polysaccharides, receptors, polynucleotides, and/or polynucleotide analogs; likewise, target molecules may be small molecules, polypeptides, proteins, cyclic polypeptides, peptidomimetics, aptamers, antibodies, scFvs, polysaccharides, receptors, polynucleotides, and/or polynucleotide analogs. Any combination of materials with specific binding properties for one another may be used as capture reagent/analyte pairs in the present invention.

As used herein, the term "small molecule" refers to compounds having molecular mass of less than 3000 Daltons, preferably less than 2000 or 1500, still more preferably less than 1000, and most preferably less than 600 Daltons. Preferably but not necessarily, a small molecule is not an oligopeptide.

As used herein, the term "polypeptide" refers to a covalent assembly comprising at least two monomeric amino acid units linked to adjacent amino acid units by amide bonds. An "oligopeptide" is a polypeptide comprising a short amino acid sequence (i.e., greater than 2 to a few hundred amino acids). An oligopeptide is generally prepared by chemical synthesis or by fragmenting a larger polypeptide. Examples of polypeptide drugs include, but are not limited to, therapeutic antibodies, insulin, parathyroid hormone, polypeptide vaccines, and antibiotics such as vancomycin. Novel polypeptide drugs may be identified by, e.g., phage display methods.

As used herein, the term "antibody" refers to an immunoglobulin molecule obtained by in vitro or in vivo generation of an immunogenic response, and includes both polyclonal, monospecific and monoclonal antibodies, and antigen binding fragments thereof (e.g., Fab fragments). An "immunogenic response" is one that results in the production of antibodies directed to one or more antigens after the appropriate cells have been contacted with such antigens.

As used herein, the term "single-chain variable region fragment" or "scFv" refers to a variable, antigen-binding determinative region of a single antibody light chain and antibody heavy chain linked together by a covalent linkage having a length sufficient to allow the light and heavy chain portions to form an antigen binding site. Such a linker may be as short as a covalent bond; preferred linkers are from 2 to 50 amino acids, and more preferably from 5 to 25 amino acids.

As used herein, the term "polynucleotide" refers to a molecule comprising a covalent assembly of nucleotides linked typically by phosphodiester bonds through the 3' and 5' hydroxyls of adjacent ribose or deoxyribose units. An "oligonucleotide" is a polynucleotide comprising a short base sequence (i.e., greater than 2 to a few hundred nucleotides, with 25- to 50-nucleotide oligomers being common). Polynucleotides include both RNA and DNA, may assume three-dimensional shapes such as hammerheads, dumbbells, etc., and may be single or double stranded. Polynucleotide drugs can include ribozymes RNAi constructs, and polynucleotide vaccines. Polynucleotides may also comprise one or more substitutions, e.g., a ribose or deoxyribose substituted at the 2' and/or 3' position with -alkyl (e.g., --O-methyl, --O-ethyl, --O-propyl), -methoxyethoxy, -allyl, -amino, or -fluoro.

As used herein, the term "polynucleotide analog" refers to a molecule that mimics the structure and function of an polynucleotide, but which is not a covalent assembly of nucleotides linked by phosphodiester bonds. Peptide nucleic acids, comprising purine and pyrimidine bases linked via a backbone linkage of N-(2-aminoethyl)-glycine units, is an example of an oligonucleotide analog.

The term "polysaccharide" as used herein refers to a carbohydrate comprising 2 or more covalently-linked saccharide units. An "oligosaccharide" is a polysaccharide comprising a short saccharide sequence (i.e., greater than 2 to several thousand saccharide units).

As used herein, the term "cyclic polypeptide" refers to a molecule comprising a covalent assembly of monomeric amino acid units, each of which is linked to at least two adjacent amino acid units by amide bonds to form a macrocycle.

As used herein, the term "peptidomimetic" refers to a molecule that mimics the structure and function of a polypeptide, but which is not a covalent assembly of amino acids linked by amide bonds. A peptoid, which is a polymer of N-substituted glycine units, is an example of a peptidomimetic.

The term "aptamer" as used herein refers to polynucleotides that bind to non-polynucleotide target molecules (e.g., a polypeptide or small molecule).

A preferred arrayed optical thin film test surface is comprised of a substrate supporting an optical thin film test surface. Preferred substrate materials include materials that are substantially rigid, such as glass, rigid plastics, metals, silicon, etc. Particularly preferred substrate materials are described hereinafter. The substrate may inherently have a reflective surface to participate in the generation of the thin film effect to be measured, or may be modified to provide such a reflective surface, e.g., by vapour deposition of a metal layer. Alternatively, in various embodiments a transmissive substrate may be preferred. In various embodiments described hereinafter, the arrayed optical thin film test surface may further comprise one or more of the following additional layers placed upon the reflective surface: an anti-reflective layer; and an attachment layer providing a covalent or non-covalent linkage to immobilize the capture reagents. As described herein, each of these layers are optional, as an anti-reflective layer is not required in all modes of the invention; and the capture reagents may be directly immobilized to sites on the surface.

A preferred format is one that places individual arrayed optical thin film test surfaces into a device readily amenable to reagent delivery and assay manipulation, either manually or using off the shelf robotics. Thus, in various embodiments, 96-position plates providing spacing of individual array locations akin to that found in commercial 96-well plates are used to house a plurality of individual arrays for use in the methods and apparatuses described herein. It should be understood that additional plate configurations are within the scope of this invention, including multiples of 96 (e.g., 384 and 1536) wells, which are conveniently used with commercially available liquid handling robots.

In additional aspects, the invention relates to methods for constructing the arrayed optical thin film test surfaces and test surface carriers. As described hereinafter, the test surface carrier of the present invention provides advantages for a wide range of detection technologies beyond the optical thin film methods.

The methods and devices described herein are particularly useful for multiplexed detection of the presence or amount of a plurality of analytes in samples. The term "analyte" or "target" refers to any molecule being detected by an assay. The analyte (or target) is typically detected by immobilizing one or more binding partners (referred to herein as "capture reagents") at a test location on an arrayed optical thin film test surface. This binding partner immobilizes the analyte for detection by the methods described herein.

Preferred are biological samples. The term "biological sample" refers to a sample obtained from an organism. Such a sample may be obtained for the purpose of diagnosis, prognosis, or evaluation of a human in a clinical setting. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred biological samples are blood samples, tissue samples, stool samples, sputum samples, serum samples, plasma samples, cerebrospinal fluid samples, urine samples, and other fluids derived from a patient, organism, or sample.

In another aspect, the present invention also relates to methods, software and associated hardware, for manual to fully automated acquisition and processing of an image acquired from an array or array of arrays. While described herein in reference to analysis of arrayed optical thin film test surfaces, the skilled artisan will understand that these methods, software, and hardware are applicable to the analysis of arrays generally, including tissue arrays (e.g., MAXARRAY.TM. commercially available from Xymed); nucleic acid arrays and microarrays (e.g., GENECHIP.RTM. commercially available from Affymetrix); protein/nucleic acid arrays and microarrays (e.g., commercially available from Panomics); antibody arrays and microarrays (e.g., commercially available from Clontech); and protein arrays and microarrays (e.g., commercially available from Ciphergen).

In preferred embodiments, an integrated "image analysis station," is provided which comprises one or more, and preferably all, of the following elements: a test surface carrier comprising a plurality of arrays as described herein; optical components for illuminating the array(s) for generation of a signal from a plurality of discretely addressable locations; the optical components comprise a diffuse light source; a digital camera for recording images of one or more arrays; optical components for focus and/or frame control of the digital camera; a stage for movement of the test surface carrier relative to the field of view of the digital camera; stage movement mechanism; a manual control for the stage movement mechanism; a manual control for the digital camera; a computer processor integrated with the stage movement mechanism and/or the digital camera; software providing instructions for predetermined stage movement and/or camera control; a digital storage medium for recording of images and results; and/or a user interface for inputting commands and/or viewing images and/or results. In preferred embodiments, image analysis is performed automatically for all reacted arrays within the test surface carrier with no user intervention.

The terms "image analysis" and "image processing" as used herein refer to acquisition of one or more digital images from an array, and the use of the image(s) collected to determine the presence or amount of one or more analytes at one, and preferably at a plurality, of discretely addressable locations on the array.

The term "optical components for illuminating the array" as used herein refers to a light source and associated optical elements for providing the desired incident light on an array. Depending on the assay format, the optical components for illuminating the array may simply be a white light or a coherent light source; or may include a filter between the light source and the array to remove undesired light wavelengths (e.g., a low-pass, high-pass, or band-pass filter); a polarizer between the light source and the array to alter the polarization state of the light; and/or other components commonly used by the artisan in reflectometry and/or ellipsometry. Ellipsometric methods may require the use of a coherent and/or monochromatic light source.

The term "diffuse light source" as used herein refers to a light source providing substantially even illumination across the field of view of the digital camera.

The term "digital camera" as used herein refers to a camera that provides a digital output signal corresponding to an image obtained by the camera. Suitable cameras, including CMOS (Complementary Metal Oxide Semiconductor, APS (active pixel sensor), CCD, and non-CCD cameras, are well known in the art. Preferred types of CCD cameras are Linear, Interline, Full-Frame, and Frame-Transfer. A Linear CCD consists of a single row of pixels; to define an image, a Linear CCD must be scanned across the plane of the image, building the picture row by row. Interline, Full-Frame, and Frame-Transfer designs are considered Area Array CCDs, because they are composed of multiple rows and columns forming a rectangular or square area. In an Interline CCD, each pixel has both a photodetector and a charge storage area. The storage area is formed by shielding or masking part of the pixel from light and using it only for the charge transfer process. Full-Frame CCDs devote the entire pixel to image capture. Therefore, when the charge transfer occurs, the pixel is busy and cannot continue to capture photons. To keep the pixels from continuing to read additional light when they are involved in charge transfer (which can lead to light smear on the image), a mechanical shutter between or behind the camera lens is often employed. Finally, Frame-Transfer CCDs are similar to Full-Frame, but they mask out half of the array to provide temporary storage for the electric charges, referred to as the "storage array". Analog cameras coupled to an analog-to-digital converter are also within the scope of the term, as such cameras provide the required digital output of images for further processing by a computer processor.

An image is said to be "recorded" by a camera if the image is acquired for processing by a computer processor. As described herein all or a portion of the image may be stored (either temporarily or permanently) within the camera electronics, or may be transferred to an attached digital storage device, or may be directly transferred to the computer processor without storage. Images may be acquired as a single test surface at a time or as multiple test surfaces within a single image, as static images or real time images, or as continuous or scanning mode images, depending on the requirements of a particular device and/or the throughput requirements.

The term "optical components for focus control" as used herein refers to optics and associated mechanical hardware employed to bring an area of interest into focus for recording by a digital camera. Similarly, "optical components for frame control" refers to optics and associated hardware employed to provide zoom and pan control to the digital camera.

The term "stage" as used herein refers to mechanical hardware required to support and provide movement along one or more axes of a test surface carrier. Preferably, a stage provides movement along orthogonal axes arbitrarily labeled X and Y; and in certain embodiments includes movement along a Z (perpendicular to the X/Y plane) axis.

The term "stage movement mechanism" as used herein refers to components (e.g., stepper motors, gearing, rack and pinion elements, bearings, etc.) providing movement to the stage.

A manual control or computer processor is "integrated" with an element of the device if instructions may be relayed from the manual control or computer processor to the element, providing a subsequent action by the element. Preferably, this integration also provides feedback from the device to the manual control or computer processor. For example, a digital camera that is integrated with a computer processor may receive instructions from the processor to record an image, and/or all or a portion of the image data may be transferred from the camera to the processor. Integration may be provided in a wired fashion (e.g., via hard wiring, a serial port (such as a standard RS-232 port), a USB port, a "fire wire" port, etc.) or wireless fashion (e.g., connected via an infrared connection, a radio frequency connection, a Bluetooth.RTM. connection, etc.).

The term "software" as used herein refers to a set of instructions, programs, and/or procedures stored in a volatile or non-volatile digital medium, for execution by a computer processor. Such software may be stored on hard or floppy disks, in volatile or non-volatile memory, on optical media, etc. In the present invention, software may provide instructions for performance of an assay, e.g., by robotic systems; for recording of digital images; and/or for analysis of digital images as described hereinafter.

The term "computer processor" refers to a digital device for performing the logic operations of a computer's program, often referred to as a "CPU." Typically, a computer processor comprises a datapath having an arithmetic logic unit (ALU) that performs arithmetic/logic operations, an address generation unit to provide memory addresses, and a control unit to provide the proper control signals for the various devices of the datapath to perform the desired operation(s). Computers typically have a processor, a main memory, a secondary storage device, and a bus for connecting the processor, the main memory and peripheral devices. Digital cameras may be connected to the computer via this bus, or via parallel or serial ports. Any of a number of well known computer processors, such as processors from Intel Corporation, of Santa Clara, Calif, may be used in the devices described herein.

The term "digital storage medium" as used herein refers to any medium in which information is stored in digital form. These include hard and floppy disks, optical disks, random access memory, read only memory, etc.

The term "user interface" as used herein refers to an element allowing user interaction with the device of the present invention, including one or more of the following: keyboards, mice, joysticks, keypads, touchscreens, monitors, etc.

Except as otherwise noted, the term "about" as used herein refers to +/-10% of any given measurement.

While the hardware of the image analysis station described herein is described in terms of the detection of thin film changes, the skilled artisan will understand that various components, including in particular the software, could be used in conjunction with any image analysis method regardless of the method of signal generation. The image merely needs to provide signals that contrast the background and possess some spatial resolution of the elements within the array that can be stored in an appropriate processing format. Thus the software is compatible with fluorescence, chemilluminescence, and other chromophores. Thus, in one aspect, the present invention relates to methods and devices, including software, for automated image analysis to determine the presence or amount of one or more analytes at one, and preferably at a plurality, of discretely addressable locations on an array.

As described herein, the image analysis instruments of the present invention preferably analyze a large number of arrays with limited user input and no user location of the array, most preferably in a completely automated manner. But manual sequences can still be performed if required or desired. The image analysis station has been selected to read a highly reflective test surface that generates signal in the reflected light from the interaction of light with a thin film generated on the surface. The thin film properties of the surface are permanent records of the reaction and can be analyzed as many times as required. The signal generated is not susceptible to photo-bleaching or photodecay and thus is stable throughout the analysis procedure. As the thin film effect is inherent in the layers of the device, there is no cross-talk between reacted zones such as can be observed when measuring fluorescence or other chromophores.

To facilitate ease of use, the image analysis station is designed to analyze surfaces that are mounted in the bottom of a depression, such as in a microtiter well. This provides an easy mechanism to manipulate and deliver samples and reagents but requires that the image analysis station reproducibly locate each well and then be capable of focusing into the well to acquire the image without interference from the walls of the wells. The same optical configuration can be used to analyze surfaces presented in a wide variety of other delivery formats including a simple slide format or individual test surfaces. The stage preferably is adaptable to hold each type of format and appropriately move the test surfaces to image each array in the device.

The test surface carrier described in the following preferred embodiments provides square wells to receive the manufactured test surfaces that are coated with an array of biological capture reagents. While commercially available microtiter plates can serve as the template for the construction of the test surface carrier, an improved plate designed for this specific application is described. This microtiter format plate design could be used to deliver any type of test surface desired but is particularly well suited for use with optical thin film surfaces. Optical thin film surfaces may be analyzed through upper surface reflection or for transmission measurements depending on the design of the optical support or surface and the optical path of the detection system. The signal generated is a function of the test surface used. For example a glass support may be used in combination with a fluorescent or chemilluminscent tag or marker. Measurements may be made in the reflectance or transmission mode. The improved test surface carrier or microtiter plate maintains the footprint of existing microtiter plates and thus is compatible with all of the off the shelf, automated, sample processing equipment. Thus the improved test surface carrier is suited to high throughput applications.

Once an array is reacted, and regardless of the method of signal generation, various image analysis tools typically employed by the artisan require the user to define or locate the position of the array, the number of elements in the array, and the location of the elements within the array. This is generally accomplished by requiring the user to select an array size and generating a grid that matches the selected parameters. The user then must drag the grid over an image of the reacted array and ensure that each grid element corresponds to an appropriate array element. Thus the user can account for any skew or stagger or mis-alignment of elements within the array. While performance of these existing tools is well suited to analysis of single arrays, even with a large number of elements, they are not well suited to analysis of large numbers of arrays, of even a limited number of elements, with any frequency of analysis. The new "spot finding" methods, provided as software or as general or special purpose computers programmed to perform the required steps of this invention, advantageously address moderate to high throughput applications.

The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the preferred embodiments, as well as from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart illustrating one embodiment of a method for generating a table of spot positions and intensities.

FIGS. 2A-2F illustrate one embodiment of a test surface carrier according to the present invention;

FIG. 3 illustrates the assembly of optical path components for an image analysis instrument according to an embodiment;

FIG. 4 illustrates an embodiment of a base support plate for the image analysis instrument;

FIGS. 5A-5D and 6A-6D illustrate embodiments of side support brackets for the image analysis instrument;

FIGS. 7A-7C illustrate an embodiment of a back support for the image analysis instrument;

FIGS. 8A-8B illustrate an embodiment of a camera support plate for the image analysis instrument;

FIGS. 9A-9C illustrate an embodiment of an extension tube bracket for the image analysis instrument;

FIGS. 10A-10B illustrate an embodiment of an extension tube clamp for the image analysis instrument;

FIGS. 11A-11C illustrate exemplary formation of spots in an arrangement according to the invention;

FIGS. 12A-12B illustrate gradients in an array of spots;

FIG. 13 illustrates a voting circle transform for gradient magnitudes and directions;

FIG. 14 illustrates exemplary CV values for the average of all spots for a set of runs;

FIG. 15 illustrates the intensity ratio for a set of runs; and

FIG. 16 illustrates the information illustrated in FIG. 15 for homozygous spots.

DETAILED DESCRIPTION

As discussed above, the present invention is described in reference to analysis of arrayed optical thin film test surfaces. The skilled artisan will understand that these methods, software, and hardware are applicable to the analysis of arrays generally. For thin film imaging, the image analysis station includes one or more, and preferably each, of the following: an optical test device; a CCD camera; a coaxial diffuse light source; an optional polarizer; an extension tube; focusing lens; optional auto-zoom and auto-focus capability; an x, y stage (with or without closed loop positional control); support structures; an instrument housing; a light power source; a stage controller; a joystick for manual stage control; a computer or equivalent for software and hardware control; a user interface; a monitor; and software.

The test surface carrier is a formatting device designed to deliver the arrayed optical thin film test surface(s), each comprising a patterned array of discrete capture locations, provide for reagent delivery and assay processing, and include a surface that will seat into the x, y stage in a stable fashion. The delivery platform can be in the shape of a standard microscope slide or configured as a standard microtiter plate of any well number. The preferred embodiment uses square wells to receive the test surface. The test surface carrier can be used to deliver test surfaces for non-thin film applications.

The optical thin film test surface may be cut, scribed, or otherwise broken into appropriate dimensions, and then glued or fused into the test surface carrier. The test surface carrier may hold one or more optical thin film test surfaces depending on the configuration of the test surface carrier. A preferred configuration for the test surface carrier is a 96 well microtiter plate having well spacings that correspond to common commercially-available microtiter plates. In the preferred embodiments, each well contains a 7 mm.times.7 mm optical thin film test surface; thus, preferably the wells in the plate are square. If other optical thin film test surface configurations are used (e.g., rectangular, circular, etc., each well may be designed appropriately to accommodate the test surface. When the microtiter plate is used to deliver test surfaces to an analytical method regardless of method of signal generation the individual array surfaces should be processed to a size that is appropriate for the dimension of the test surface carrier.

If a microscope slide is the test surface carrier then a single 1''.times.3'' test surface may be mounted in the test device or a number of smaller strips or squares may be mounted into the test carrier that retains the 1''.times.3'' size.

Regardless of the assay format or detection means used, test surfaces are prepared by immobilizing an array of capture reagents on the test surface. The capture reagents can be a complex combination of materials designed to interrogate a sample for a range of different analytes, genes, gene products, genomic DNA, small molecules, SNPS, or other materials of interest. Each capture reagent can specifically capture a target nucleic acid sequence, protein, sugar, lipid, hormone, or other analyte. As an example the capture reagents could be antibodies specific to a range of cytokines in a sample. Or the capture reagents could be a set of oligonucleotides that are specific to a combination of genes that are markers or indications of a specific disease. Or the capture reagents could be a set of oligonucleotides that are specific to SNP mutations in a specific gene that indicate the carrier status of an individual for a given disease, like cystic fibrosis (CF). Or the capture reagents can be antibodies specific to a panel of allergans for allergy screening, or specific to bacterial and/or viral antigens (or oligonucleotides for genes) for differential diagnosis of the caustive agent of infections like a respiratory infection.

The software designed for the image analysis of individual arrays automatically identifies and verifies the location and signal strength for each test element in the array. To automatically process the array, the preferred embodiment first locates the edges of the test surface in the image if possible. If the edges are found, the area of the image outside of the edges is eliminated from further consideration. Next, any possible features of the correct size, shape and intensity are located in the image. The number of such features is counted. The count of such features is used to determine the type of sample in the image. For example, a reacted patient sample would have a feature count within a certain range; a blank would have a different range; an empty well would have yet another typical range for the count. Once the type of sample has been determined, a set of heuristic rules is applied to eliminate spurious noise features. For example, a spot within a small number of pixels of the test surface edge is more likely to be due to washing problems than actual reactions and may be eliminated from further consideration. Next, the algorithm attempts to locate the grid structure from the remaining spot locations. A clustering algorithm and additional heuristics may be applied to define the location of the rows and columns of the spot grid. Rows and columns containing few or no reacted zones may have their positions estimated from the positions and spacings of other rows and columns containing more spots. Finally, spots are matched to locations in the grid and signals are measured. A table of spot positions and signal strengths is generated for further classification. The software and the test surface carrier to be described are generally suited for use in non-thin film applications as well. One embodiment of a method for generating the table for spot positions and signals is described below with reference to FIG. 1.

Software requirements for the system include an image acquisition protocol, stage movement and positional control, array identification, array element identification, array element quantification, array corrections for skew, production of a results table by array element, background acquisition, background correction, data processing and interpretation, and result reporting. A number of commercial sources for image acquisition and stage control are available. The suppliers of CCD cameras and the stages also provide software that can be used for these functions and then integrated into a final software package. Scanalytics provides an image analysis package that integrates image acquisition and stage control with other software protocols for the collection and analysis of an imaged array. However, this software requires a large amount of user interaction to analyze even small arrays.

Test Surface Design

Methods for the design of optical thin film test surfaces for use in optical thin film assays are well known in the art. See, e.g., U.S. Pat. No. 5,629,214, which is hereby incorporated in its entirety, including all tables, figures and claims. A wide range of rigid materials may form the substrate, including glass, fused silica, plastic, ceramic, metal, and semiconductor materials. The substrate may be of any thickness desired. Flexible optical substrates include thin sheets of plastic and like materia