WO2006017642A2 - Filtrage de signaux pendant un scan de matrice - Google Patents

Filtrage de signaux pendant un scan de matrice Download PDF

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Publication number
WO2006017642A2
WO2006017642A2 PCT/US2005/027693 US2005027693W WO2006017642A2 WO 2006017642 A2 WO2006017642 A2 WO 2006017642A2 US 2005027693 W US2005027693 W US 2005027693W WO 2006017642 A2 WO2006017642 A2 WO 2006017642A2
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WO
WIPO (PCT)
Prior art keywords
signal
pixel
signals
array
digital signals
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PCT/US2005/027693
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English (en)
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WO2006017642A3 (fr
Inventor
Kenneth L. Staton
John F. Corson
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Agilent Technologies, Inc.
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Publication date
Priority claimed from US10/912,463 external-priority patent/US7818129B2/en
Priority claimed from US10/912,427 external-priority patent/US7876962B2/en
Priority claimed from US10/912,661 external-priority patent/US7877212B2/en
Priority claimed from US10/912,027 external-priority patent/US7627435B2/en
Application filed by Agilent Technologies, Inc. filed Critical Agilent Technologies, Inc.
Publication of WO2006017642A2 publication Critical patent/WO2006017642A2/fr
Publication of WO2006017642A3 publication Critical patent/WO2006017642A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/10—Scanning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30072—Microarray; Biochip, DNA array; Well plate

Definitions

  • Arrays of surface-bound binding agents may be used to detect the presence of particular targets, e.g., biopolymers, in solution.
  • the surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules in solution.
  • binding interactions are the basis for many of the methods and devices used in a variety of different fields, e.g., genomics (in sequencing by hybridization, SNP detection, differential gene expression analysis, identification of novel genes, gene mapping, finger printing, etc.) and proteomics.
  • One typical array assay method involves biopolymeric probes immobilized in an array on a substrate, such as a glass substrate or the like.
  • a solution containing analytes that bind with the attached probes is placed in contact with the array substrate, covered with another substrate such as a coverslip or the like to form an assay area and placed in an environmentally controlled chamber such as an incubator or the like.
  • the targets in the solution bind to the complementary probes on the substrate to form a binding complex.
  • the pattern of binding by target molecules to biopolymer probe features or spots on the substrate produces a pattern on the surface of the substrate and provides desired information about the sample.
  • the target molecules are labeled with a detectable tag such as a fluorescent tag or chemi luminescent tag.
  • the resultant binding interaction or complexes of binding pairs are then detected and read or interrogated, for example by optical means, although other methods may also be used.
  • optical means for example, laser light may be used to excite fluorescent tags, generating a signal only in those spots on the biochip that have a target molecule and thus a fluorescent tag bound to a probe molecule.
  • This pattern may then be digitally scanned for computer analysis.
  • optical scanners play an important role in many array-based applications.
  • Optical scanners act like a large field fluorescence microscope in which the fluorescent pattern caused by binding of labeled molecules on the array surface is scanned. In this way, a laser induced fluorescence scanner provides for analyzing large numbers of different target molecules of interest, e.g., genes/mutations/alleles, in a biological sample.
  • a detector For each pixel of a scan, a detector (e.g., a light detector such as a photomultiplier tube) typically detects light emitted from the surface of a microarray, and outputs an analog signal line that changes in amplitude according to the amount of emitted light entering the detector.
  • This analog signal is usually sampled and digitized using an analog-to-digital converter (AfD converter) and integrated using a digital signal processor (DSP) to provide data, e.g., a numerical evaluation of the brightness of the pixel. This data is usually stored and analyzed at a later date.
  • AfD converter analog-to-digital converter
  • DSP digital signal processor
  • pixel signal "noise” i.e., signals not related to the detected optical signal, for example electrical noise
  • This noise may be caused by other electronic circuitry, an electromagnetic disturbance, fluctuations in the intensity of the light used to excite the fluorescence, or a software or hardware error, for example.
  • Pixel signals containing noise are typically digitized and integrated using similar methods to those for other signals, and, as such, inaccurate data may be produced from pixels signals containing signal noise.
  • the present invention meets this, and other, needs.
  • Literature of interest includes: published U.S. Patent Applications: 20030168579,
  • the invention provides methods of evaluating a signal produced during scanning of a chemical array, comprising: obtaining digital signals for a plurality of points across a region of an array substrate and identifying and extracting data from the digital signals corresponding to feature information.
  • the method comprises saving data relating to the signals in a memory.
  • the digital signals include signals corresponding to light emitted from the surface of the chemical array.
  • signals unrelated to chromophores (or other labels) bound to the surface of the array are removed from the obtained digital signals.
  • signals unrelated to chromophores bound to features on the surface of the array are removed from the obtained digital signals.
  • the signal is sampled a plurality of times for one or more of the plurality of points.
  • the method further comprises providing an output signal representing the plurality of sampling steps.
  • the invention provides systems for performing any of the methods described above, comprising a means for obtaining digital signals and for identifying and extracting data from the digital signals corresponding to feature information.
  • a system comprises a memory for storing data relating to the signals.
  • the system comprises an array scanner comprising, or in communication with, a processor comprising the member and/or the means for obtaining and/or for identifying and/or extracting data.
  • the invention provides computer program products comprising instructions for implementing methods as described herein and/or for executing system functions as described herein.
  • an analog signal for a partially saturated pixel is sampled to obtain a set of non-saturated digital signals and a set of saturated digital signals.
  • the saturated digital signals are then processed to produce data for the pixel.
  • the invention provides a method for producing data for a partially saturated pixel during scanning of a chemical array.
  • This method generally includes: sampling an analog signal for a partially saturated pixel to provide a set of non- saturated digital signals and a set of saturated digital signals; and processing said non- saturated signals to produce data for the partially saturated pixel.
  • the method may comprise estimating said saturated digital signals by extrapolating said non-saturated digital signals to provide a set of estimated digital signals
  • the analog signal for a partially saturated pixel is sampled at least about 10 times.
  • the method further involves employing a current to voltage converter that converts an analog current representing intensity of said pixel to an analog voltage signal.
  • the method may also comprise tagging data corresponding to the partially saturated pixel with an identifier to indicate that the data is derived from a partially saturated pixel.
  • the methods involve producing at least two analog signals for a pixel using a multi-gain signal detection system, integrating at least one of these signals, and outputting a single integrated signal representing the pixel.
  • the methods involve detecting, integrating and outputting a signal for a non- saturated input signal for the pixel.
  • the invention also provides a method for evaluating a pixel during scanning of a chemical array.
  • this methods includes: producing a plurality of analog signals for said pixel using a multi-gain signal detection system; identifying a non-saturated analog signal for the pixel; and integrating the non-saturated analog signal for said signal, to evaluate the pixel.
  • the plurality of signals may be a plurality of voltage or current signals.
  • the subject method for evaluating a pixel includes: providing for detection of a set of non-saturated digital signals for said pixel; integrating a set of digital signal that is non-saturated; and outputting a single integrated signal for said set of digital signals that are not saturated.
  • the single integrated signal is data
  • said method further comprises storing the data on a computer-readable medium.
  • methods for identifying feature boundary pixels involve evaluating a pixel signal to identify any difference in amplitude between a first part of the signal and a second part of the signal. If the difference is significant, the pixel signal may be indicated as a pixel representing a feature boundary. Such a pixel may be used to identify the perimeter of features.
  • the invention provides a method of determining whether a pixel signal produced during scanning of a chemical array (e.g., a nucleic acid or polypeptide array) is a feature boundary pixel signal, comprising: evaluating change in amplitude of said pixel signal (to provide, in certain embodiments a numerical evaluation); wherein a pixel signal having a significant change in amplitude is a feature boundary pixel signal.
  • Certain embodiments of this method comprise evaluating any difference in amplitude between a first part of the signal and a second part of the signal to provide an evaluation of amplitude change of the pixel.
  • a pixel signal having a change in amplitude above a pre-determined level indicates that the signal is a feature boundary pixel signal.
  • a pixel signal having a line of best fit having a slope greater than a threshold slope indicates that said pixel is a feature boundary pixel.
  • the method may comprise evaluating closeness of the pixel signal to its line of best fit.
  • Any evaluation produced by the subject method may be stored on a computer readable medium.
  • the method may further comprise integrating the pixel signal to produce, for example, a numerical evaluation of the pixel. Such a numerical evaluation may be stored on a computer a readable medium.
  • the numerical evaluation of the pixel is adjusted if the pixel is a feature boundary pixel, or, in alternative embodiments, the numerical evaluation of the pixel is annotated with information indicating an adjustment factor for adjusting the numerical evaluation if the pixel is a feature boundary pixel.
  • the method further comprises indicating which end of the pixel signal represents signal from a feature area or which end of said pixel signal represents signal from a non-feature area if said pixel is a feature boundary pixel.
  • the method further comprises indicating where in the pixel signal transition between signal from a feature area and signal from a non-feature area occurs if the pixel is a feature boundary pixel.
  • the method may comprise scanning a chemical array to provide the pixel signal.
  • the invention provides method of evaluating a feature on a surface of a chemical array, comprising: scanning the feature to provide a plurality of pixel signals for the feature; integrating pixel signals to provide numerical evaluations of the pixels; and adjusting the numerical evaluations using the adjustment factor discussed above.
  • Additional subject methods according to aspects of the invention comprise identifying a set of conformant digital signals for a pixel, and integrating those signals.
  • the non-conformant signals i.e., the signals that correspond to undesirable signal noise, are generally filtered out prior to integration of the pixel signal.
  • the resultant numerical evaluation is more accurate than if the methods are not employed.
  • the invention provides a method of evaluating a signal for a pixel produced during scanning of a chemical array (e.g., a polypeptide or nucleic acid array). This method may involve providing a set of conformant digital signals for said pixel signal; and integrating the conformant digital signals, to evaluate the pixel signal.
  • a chemical array e.g., a polypeptide or nucleic acid array
  • the conformant digital signals are produced by filtering signals in a time domain and in certain embodiments, the conformant digital signals are produced by filtering signals in a frequency domain.
  • the conformant digital signals are produced by: Fourier transforming the time samples of said pixel to produce a plurality of frequency components, reducing the magnitude of frequency components above a threshold as a function of their frequency, and reverse Fourier transforming the adjusted frequency components.
  • the method may produce data (i.e., a numerical evaluation) for a pixel that may be output from a signal processor and, in certain embodiments, stored on a computer readable medium.
  • Other embodiments of the method may employ a filter to filter out any non- conformant digital signals prior to signal integration, or an algorithm to identify the conformant digital signals. These embodiments may involve: producing an analog signal for a pixel; digitizing the analog signal to provide a plurality of digital signals for said pixel; identifying a set of conformant digital signals from said plurality of digital signals; and integrating the conformant digital signals.
  • the invention provides a system for evaluating a pixel signal produced during scanning a chemical array.
  • the system generally comprises: a) a multi-gain detection system that produces a plurality of analog signals representative of a pixel; b) a converter for digitizing the plurality of analog signals to produce a set of digital signals for each analog signal; and, c) a signal processor that: i) identifies a set of non-saturated digital signals for the pixel, and ii) integrates the set of non-saturated digital signals for said pixel, to evaluate the pixel.
  • the system comprises one or more of a photodetector (e.g., a PMT or the like), a single multi-gain detector, a plurality of detectors each having a different gain setting, a plurality of current-to- voltage converters or a multi-gain voltage amplifier.
  • the converter comprises a plurality of analog-to-digital converters.
  • the invention provides a chemical array scanner.
  • the scanner includes: a laser excitation system; a detection system that produces an analog signal representative of emitted light from the surface of an array; a system for performing the above method; and a storage medium for storing data produced by the method.
  • the scanner may contain an analog-to-digital signal converter; and a digital signal processor for integrating the non-saturated and the estimated digital signals.
  • the storage medium may be computer memory.
  • the invention provides a chemical array scanner comprising a laser excitation system; a detection system that produces an signal representative of emitted light from the surface of an array; and a system for performing the above method.
  • the scanner may contain an analog-to-digital converter; and a signal processor programmed to perform the above method.
  • the scanner may contain a storage medium, e.g., computer memory, for storing data.
  • the invention also provides a computer-readable medium for evaluating a pixel signal produced during scanning a chemical array according to any of the methods described above.
  • the computer-readable medium comprises: programming products for execution by a digital signal processor to produce data for a pixel represented by saturated and non-saturated digital signals, the programming comprising: instructions for estimating the saturated digital signals by extrapolating the non-saturated digital signals; and instructions for integrating the estimated digital signals from the non-saturated digital signals to produce data for the pixel.
  • the computer-readable medium may further comprise instructions for executing the programming when a partially-saturated pixel is detected.
  • the output of the programming may be data for a partially saturated pixel.
  • the output may be tagged to indicate that data is estimated from a set of saturated and non-saturated digital signals.
  • the invention also provides a computer-readable medium that contains programming for execution by a digital signal processor to produce data for a pixel represented by multiple digital signals or sets of digital signals of varying magnitude, the programming including: instructions for integrating a set of non-saturated digital signals for said pixel to produce an integrated signal representing said signal; and outputting said integrated signal to produce data for said pixel.
  • the computer- readable medium further includes instructions for executing the programming when a non- saturated signal for said pixel is detected.
  • programming provides for tagging the output of the processor to indicate that the data is from a saturated pixel.
  • the invention also provides a computer-readable medium comprising: programming for execution by a digital signal processor to produce data for a pixel in a scan of a chemical array, comprising: instructions for evaluating change in amplitude of said pixel signal, to produce data for said pixel.
  • the computer readable medium may also comprises programming to integrate said pixel signal.
  • the computer-readable medium may provide for a signal processor output that is a multi-bit code, in which a first part of said code represents an evaluation of amplitude any amplitude change of said signal and a second part of said code represents said integrated signal.
  • the invention provides a computer-readable medium comprising: programming products for execution by a digital signal processor to produce data for a pixel, the programming comprising: instructions for identifying a set of conformant digital signals from a pixel signal; and instructions for integrating the conformant digital signals to produce data for the pixel.
  • the computer-readable medium may further comprise instructions for executing the programming products when a pixel signal containing a non-conformant signal is detected.
  • the invention further provides a processor or computer containing any one or more of above computer-readable media and a chemical array scanner comprising or, in communication with, that processor and/or computer.
  • the invention provides a method of assaying a sample which includes: (a) contacting the sample with a chemical array of two or more chemical ligands immobilized on a surface of a solid support; and (b) reading the array with the above- described chemical array scanner to obtain data.
  • the reading step of this method may include: producing a plurality of analog signals for a pixel using a multi-gain signal detection system to produce sets of digital signals for the pixel; identifying a set of non-saturated analog signals for the pixel; and integrating the non- saturated analog signal for said signal, to produce data for said pixel.
  • the invention also provides a method of assaying a sample.
  • the method comprises: (a)contacting the sample with a chemical array of two or more chemical ligands immobilized on a surface of a solid support; and (b) reading the array with a chemical array scanner according to the above to obtain data.
  • Step (b) in this method may include: sampling a partially saturated analog signal to provide a set of non-saturated digital signals and a set of saturated digital signals for said partially saturated pixel; and processing the non-saturated signals to produce data for a partially saturated pixel.
  • Step (b) of this method may comprise estimating the saturated digital signals by extrapolating the non-saturated digital signals to provide a set of estimated digital signals
  • the invention also provides a method of assaying a sample which comprises: contacting a sample with a chemical array of two or more features immobilized on a surface of a solid support; reading the array with a chemical array scanner and evaluating a a pixel signal produced during scanning a chemical array according to any of the methods and/or computer programs described above.
  • the method is used to obtain data relating to a feature boundary pixel signal as described above.
  • the invention provides a method of assaying a sample, comprising: (a) contacting said sample with a chemical array (e.g., a polypeptide or nucleic acid array) of two or more chemical ligands immobilized on a surface of a solid support; and (b) reading the array with a chemical array scanner according to the above to obtain data.
  • a chemical array e.g., a polypeptide or nucleic acid array
  • This method may include: identifying a set of conformant digital signals from a plurality of digital signals for a pixel; and integrating the conformant digital signals to produce data for the pixel.
  • the invention also provides a method including transmitting a result obtained from any of the above-described methods from a first location to a remote location, and a method including receiving data representing data obtained by any of the above-described methods.
  • the invention also provides a kit for use in a chemical array optical scanner, comprising a computer-readable medium according to the above; and at least one chemical array.
  • the chemical array may be a biopolymer array, e.g., such as a nucleic acid or polypeptide array.
  • Fig. 1 schematically illustrates aspects of an embodiment of the invention described herein.
  • Fig. 2 schematically illustrates aspects of another embodiment of the invention described herein.
  • Fig. 3 schematically illustrates an apparatus as may be used in the present invention.
  • Fig. 4 schematically illustrates aspects of another embodiment of the invention described herein.
  • Fig. 5 schematically illustrates aspects of multi-gain detection systems described herein.
  • Fig. 6 shows three panels of figures, A, B and C, and schematically illustrates aspects of multi-gain detection systems described herein.
  • Fig. 7 schematically illustrates one embodiment of the signal acquisition, processing and integration methods described herein.
  • Fig. 8 schematically illustrates one example of a signal integration method that may be used in the subject methods.
  • Fig. 9 schematically illustrates a system for performing the subject methods.
  • a "biopolymer” is a polymer of one or more types of repeating units. Biopolymers are typically found in biological systems and particularly include polysaccharides (such as carbohydrates), polypeptides (which term is used to include peptides and proteins) and polynucleotides as well as their analogs such as those compounds composed of or containing amino acid analogs or non-amino acid groups, or nucleotide analogs or non- nucleotide groups.
  • Biopolymers include polynucleotides in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone, and nucleic acids (or synthetic or naturally occurring analogs) in which one or more of the conventional bases has been replaced with a group (natural or synthetic) capable of participating in Watson-Crick type hydrogen bonding interactions.
  • Polynucleotides include single or multiple stranded configurations, where one or more of the strands may or may not be completely aligned with another.
  • nucleotide refers to a sub-unit of a nucleic acid and has a phosphate group, a 5 carbon sugar and a nitrogen containing base, as well as functional analogs (whether synthetic or naturally occurring) of such sub-units which in the polymer form (as a polynucleotide) can hybridize with naturally occurring polynucleotides in a sequence specific manner analogous to that of two naturally occurring polynucleotides.
  • Biopolymers include DNA (including cDNA), RNA, oligonucleotides, and PNA and other polynucleotides as described in U.S. Patent No.
  • oligonucleotide generally refers to a nucleotide multimer of about 10 to 100 nucleotides in length, while a “polynucleotide” includes a nucleotide multimer having any number of nucleotides.
  • a "biomonomer” references a single unit, which can be linked with the same or other biomonomers to form a biopolymer (e.g., a single amino acid or nucleotide with two linking groups one or both of which may have removable protecting groups).
  • An "array,” or “chemical array” includes any two-dimensional or substantially two- dimensional (as well as a three-dimensional) arrangement of addressable regions bearing a particular chemical moiety or moieties (e.g., biopolymers such as polynucleotide or oligonucleotide sequences (nucleic acids), polypeptides (e.g., proteins), carbohydrates, lipids, etc.) associated with that region.
  • the preferred arrays are arrays of polymeric binding agents, where the polymeric binding agents may be any of: polypeptides, proteins, nucleic acids, polysaccharides, synthetic mimetics of such biopolymeric binding agents, etc.
  • the arrays are arrays of nucleic acids, including oligonucleotides, polynucleotides, cDNAs, mRNAs, synthetic mimetics thereof, and the like.
  • the nucleic acids may be covalently attached to the arrays at any point along the nucleic acid chain, but are generally attached at one of their termini (e.g. the 3' or 5' terminus).
  • the arrays are arrays of polypeptides, e.g., proteins or fragments thereof.
  • Any given substrate may carry one, two, four or more or more arrays disposed on a front surface of the substrate.
  • any or all of the arrays may be the same or different from one another and each may contain multiple spots or features.
  • a typical array may contain more than ten, more than one hundred, more than one thousand more ten thousand features, or even more than one hundred thousand features, in an area of less than 20 cm or even less than 10 cm .
  • features may have widths (that is, diameter, for a round spot) in the range from a 10 ⁇ m to 1.0 cm.
  • each feature may have a width in the range of 1.0 ⁇ m to 1.0 mm, usually 5.0 ⁇ m to 500 ⁇ m, and more usually 10 ⁇ m to 200 ⁇ m.
  • Non-round features may have area ranges equivalent to that of circular features with the foregoing width (diameter) ranges.
  • At least some, or all, of the features are of different compositions (for example, when any repeats of each feature composition are excluded the remaining features may account for at least 5%, 10%, or 20% of the total number of features).
  • Interfeature areas will typically (but not essentially) be present which do not carry any polynucleotide (or other biopolymer or chemical moiety of a type of which the features are composed).
  • interfeature areas typically will be present where the arrays are formed by processes involving drop deposition of reagents but may not be present when, for example, photolithographic array fabrication processes are used,. It will be appreciated though, that the interfeature areas, when present, could be of various sizes and configurations.
  • Each array may cover an area of less than 100 cm 2 , or even less than 50 cm 2 , 10 cm 2 or 1 cm 2 .
  • the substrate carrying the one or more arrays will be shaped generally as a rectangular solid (although other shapes are possible), having a length of more than 4 mm and less than 1 m, usually more than 4 mm and less than 600 mm, more usually less than 400 mm; a width of more than 4 mm and less than 1 m, usually less than 500 mm and more usually less than 400 mm; and a thickness of more than 0.01 mm and less than 5.0 mm, usually more than 0.1 mm and less than 2 mm and more usually more than 0.2 and less than 1 mm.
  • the substrate may be of a material that emits low fluorescence upon illumination with the excitation light. Additionally in this situation, the substrate may be relatively transparent to reduce the absorption of the incident illuminating laser light and subsequent heating if the focused laser beam travels too slowly over a region. For example, substrate 10 may transmit at least 20%, or 50% (or even at least 70%, 90%, or 95%), of the illuminating light incident on the front as may be measured across the entire integrated spectrum of such illuminating light or alternatively at 532 nm or 633 nm.
  • Arrays can be fabricated using drop deposition from pulse jets of either polynucleotide precursor units (such as monomers) in the case of in situ fabrication, or the previously obtained polynucleotide.
  • polynucleotide precursor units such as monomers
  • Such methods are described in detail in, for example, the previously cited references including US 6,242,266, US 6,232,072, US 6,180,351, US 6,171,797, US 6,323,043, U.S. Patent Application Serial No. 09/302,898 filed April 30, 1999 by Caren et al., and the references cited therein. As already mentioned, these references are incorporated herein by reference.
  • Other drop deposition methods can be used for fabrication, as previously described herein.
  • photolithographic array fabrication methods may be used such as described in US 5,599,695, US 5,753,788, and US 6,329,143. Interfeature areas need not be present particularly when the arrays are made by photolithographic methods as described in those patents.
  • An array is "addressable" when it has multiple regions of different moieties (e.g., different polynucleotide sequences) such that a region (i.e., a "feature” or “spot” of the array) at a particular predetermined location (i.e., an "address" on the array will detect a particular target or class of targets (although a feature may incidentally detect non-targets of that feature).
  • Array features are typically, but need not be, separated by intervening spaces.
  • the "target” will be referenced as a moiety in a mobile phase (typically fluid), to be detected by probes ("target probes”) which are bound to the substrate at the various regions.
  • a “scan region” refers to a contiguous (preferably, rectangular) area in which the array spots or features of interest, as defined above, are found. The scan region is that portion of the total area illuminated from which the resulting fluorescence is detected and recorded. For the purposes of this invention, the scan region includes the entire area of the slide scanned in each pass of the lens, between the first feature of interest, and the last feature of interest, even if there exist intervening areas which lack features of interest.
  • array layout refers to one or more characteristics of the features, such as feature positioning on the substrate, one or more feature dimensions, and an indication of a moiety at a given location.
  • “Hybridizing” and “binding”, with respect to polynucleotides, are used interchangeably.
  • remote location it is meant a location other than the location at which the array is present and hybridization occurs.
  • a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc.
  • office, lab, etc. another location in the same city
  • another location in a different city another location in a different state
  • another location in a different country etc.
  • “Communicating” information references transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network).
  • a device is “in communication with” another device, the devices are capable of transmitting or data or instructions to each other. Such devices may be networked to each other.
  • “Forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data.
  • An array “package” may be the array plus only a substrate on which the array is deposited, although the package may include other features (such as a housing with a chamber).
  • a “chamber” references an enclosed volume (although a chamber may be accessible through one or more ports). It will also be appreciated that throughout the present application, that words such as “top,” “upper,” and “lower” are used in a relative sense only.
  • a "scanner” is device for evaluating arrays.
  • an optical light source particularly a laser light source, generates a collimated beam.
  • the collimated beam is focused on the array and sequentially illuminates small surface regions of known location (i.e. a position) on an array substrate.
  • the resulting signals from the surface regions are collected either confocally (employing the same lens used to focus the light onto the array) or off-axis (using a separate lens positioned to one side of the lens used to focus the onto the array).
  • the collected signals are then transmitted through appropriate spectral filters, to an optical detector.
  • a recording device such as a computer memory, records the detected signals and builds up a raster scan file of intensities as a function of position, or time as it relates to the position.
  • intensities as a function of position, are typically referred to in the art as "pixels".
  • Arrays are often scanned and/or scan results are often represented at 5 or 10 micron pixel resolution.
  • components such as the lasers must be set and maintained with particular alignment.
  • Scanners may be bi-directional, or unidirectional, as is known in the art.
  • the scanner typically used for the evaluation of arrays includes a scanning fluorometer.
  • a number of different types of such devices are commercially available from different sources, such as such as Perkin-Elmer, Agilent, or Axon Instruments, etc., and examples of typical scanners are described in U.S. Patent Nos: 5,091,652; 5,760,951, 6,320,196 and 6,355,934.
  • assessing and “evaluating” are used interchangeably to refer to any form of measurement, and includes determining if an element is present or not.
  • the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of includes determining the amount of something present, as well as determining whether it is present or absent.
  • the term “evaluating a pixel” and grammatical equivalents thereof, are used to refer to measuring the strength, e.g., magnitude, of pixel signal to determine the brightness of a corresponding area present on the surface of an object scanned.
  • a "processor” references any hardware and/or software combination which will perform the functions required of it.
  • any processor herein may be a programmable digital microprocessor such as available in the form of a electronic controller, mainframe, server or personal computer (desktop or portable).
  • suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based).
  • a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.
  • a processor may be a "signal processor", where a signal processor receives input signals and processes those signals.
  • a signal processor may programmed or hard wired to perform one or more mathematical functions, such as those described below.
  • a signal processor may "integrate" a set of digital signals (e.g., a set of digital signals representing an analog signal or a digitized version of an analog signal).
  • integrating is meant that a set of digital signals is input into a signal processor and the signal processor provides an output signal, in certain embodiments a single output signal, that represents the set of input signals.
  • the input set of digital signals may be integrated by summing the set of input signals, however, other means for integrating (e.g., averaging, etc.) are well known in the art.
  • an analog signal is referred to as being integrated, then it is understood that the analog signal is first digitized (i.e., sampled) prior to integration. For example, if an analog signal for a pixel is to be integrated, the signal is first sampled and digitized to provide a set of digital signals, and those digital signals are integrated by a signal processor to provide an output signal, typically a binary signal, that represents a numerical evaluation of the overall magnitude of the input set of digital signals (thereby providing a numerical evaluation of the magnitude of the analog signal for the pixel).
  • the output of a signal processor may be referred herein as "data", and may be stored in memory.
  • a digital signal may be a "conformant” or “non-conformant” signal based on whether the signal has a magnitude that is above or below a threshold magnitude.
  • a “conformant” signal has a magnitude within a range defined by the threshold magnitude and a “non-conformant” signal has a magnitude outside of the range defined by the threshold magnitude.
  • Non-conformant signals frequently result from system noise (e.g., electrical noise) not related to hybridization events (e.g., non ⁇ specific hybridization or the like).
  • “non-conformant signals” are “outlier" signals that do not conform to a pre-determined or normal signal range.
  • Data from reading an array may be raw data (such as fluorescence intensity readings for each feature in one or more color channels, or, for example, the output of a signal processor that has integrated a set of digital signals for a pixel) or may be processed data such as obtained by rejecting a reading for a feature which is below a predetermined threshold and/or forming conclusions based on the pattern read from the array (such as whether or not a particular target sequence may have been present in the sample).
  • the data obtained from an array reading may be forwarded (such as by communication) to a remote location if desired, and received there for further use (such as further processing).
  • the subject methods may include a step of transmitting data from at least one of the detecting and deriving steps, to a remote location.
  • the data may be transmitted to the remote location for further evaluation and/or use.
  • Any convenient telecommunications means may be employed for transmitting the data, e.g., facsimile, modem, internet, etc.
  • a set of digital signals for a pixel may be
  • saturated “saturated”, “partially-saturated” or “non-saturated” depending on the number of saturated digital signals within the set.
  • the digital signals in a saturated set of digital signals are all saturated, none of the digital signals in a non-saturated set of digital signals are saturated, and some but not all of the digital signals within a partially-saturated set of digital signals are saturated.
  • Saturated digital signals may be identified by virtue of the fact that they are at maximal magnitude, and non-saturated digital signals may be identified by virtue of the fact that they are below maximal magnitude.
  • the methods provide "a set of non-saturated digital signals” and a "set of saturated digital signals”, and processes the non-saturated digital signals to provide an evaluation of a pixel.
  • the methods provide a means for distinguishing saturated and non-saturated signals, allowing only the set of non-saturated signals to be processed to produce an evaluation of a pixel. This is in contrast to prior art devices that do not discriminate between saturated and non-saturated signals, which are all processed be processed to produce an evaluation of a pixel.
  • a "feature boundary pixel signal” is the signal of a pixel representing a feature boundary, e.g., a signal of a pixel that transitions the boundary between a feature area and a non-feature area of an array.
  • data obtained from a feature boundary pixel signal may be associated with an "adjustment factor", where an “adjustment factor” is a value by which the data may be adjusted (e.g., multiplied by or added to) to provide a more accurate evaluation of the pixel.
  • an adjustment factor represents the difference between data produced by integrating a pixel signal that is part feature signal and part non-feature signal, and data produced by integrating the same pixel signal, correcting for the reduced magnitude of the non-feature signal.
  • a numerical evaluation of a pixel signal that is 50% feature signal and 50% non-feature signal may be adjusted (e.g., multiplied) by an adjustment factor of 2 to provide a more accurate evaluation of the pixel.
  • a numerical evaluation of a pixel signal that is 25% feature signal and 75% non-feature signal may be adjusted (e.g., multiplied) by an adjustment factor of 4 to provide a more accurate evaluation of the pixel.
  • the term "using" has its conventional meaning, and, as such, means employing, e.g. putting into service, a method or composition to attain an end. For example, if a program is used to create a file, a program is executed to make a file, the file usually being the output of the program. In another example, if a computer file is used, it is usually accessed, read, and the information stored in the file employed to attain an end. Similarly if a unique identifier, e.g., a barcode is used, the unique identifier is usually read to identify, for example, an object or file associated with the unique identifier.
  • a unique identifier e.g., a barcode
  • an analog signal for a partially saturated pixel is sampled to obtain a set of non-saturated digital signals and a set of saturated digital signals.
  • the saturated digital signals are then processed to produce data for the pixel.
  • the methods involve producing at least two analog signals for a pixel using a multi-gain signal detection system, integrating at least one of these signals, and outputting a single integrated signal representing the pixel.
  • the methods involve detecting, integrating and outputting a signal for a non- saturated input signal for the pixel.
  • methods for identifying feature boundary pixels involve evaluating a pixel signal to identify any difference in amplitude between a first part of the signal a second part of the signal. If the difference is significant, the pixel signal may be indicated as a pixel representing a feature boundary. Such a pixel may be used to identify the perimeter of features. Also provided are systems and programming for performing the subject methods, and an array scanner containing these systems and programming. The methods finds use in a variety of different applications, including both genomics and proteomics applications.
  • methods involve identifying a set of conformant digital signals for a pixel, and integrating those signals.
  • the non-conformant signals i.e., the signals that correspond to undesirable signal noise, are generally filtered out prior to integration of the pixel signal.
  • the resultant numerical evaluation is more accurate than if the methods are not employed.
  • the invention provides a method for evaluating a pixel signal produced during scanning of a chemical array.
  • the method comprises evaluating (e.g., producing a numerical evaluation of the brightness of) a partially saturated pixel, where a "partially saturated pixel" is a pixel represented by a signal that is at least partly saturated, e.g., at least one of the digital samples of the signal is at a maximum amplitude.
  • partially saturated pixels correspond to bright areas of a scan of the surface of an object, e.g., a chemical array.
  • such a signal is generated by a light detector and is an analog signal that changes in amplitude proportionally to the amount of light entering the detector.
  • the detector is a photomultiplier tube (PMT).
  • the signal may be an electrical signal and either the current or the voltage of the signal may vary in amplitude proportionally to the amount of light entering the detector. In certain embodiments it is the current of the signal that varies in amplitude according to the amount of light detected by the detector, and a "current-to-voltage" converter may be employed to convert this current signal to a voltage signal.
  • FIG. 7 plot signal amplitude (i) versus time (t) for a pixel, which, in this figure, is period of time that a light signal is detected.
  • Graph 2 shows an exemplary light signal 6, relative to the maximum intensity of light detectable by the detection system 4 (including a detector and, in certain embodiments other circuitry such as a current-to- voltage converter and/or a voltage amplifier). Some of the light signal is above the detection limit of the detection system. This light enters a detection system, and the detection system outputs an analog signal 10, as shown in graph 8. As noted in graph 8, part of the analog signal is maximal and "flat", indicating that the signal is saturated.
  • the signal output of the detection system is partially saturated (i.e. saturated during a portion of the total pixel signal time).
  • Saturated signals may be produced by any detection system component, e.g., a detector, a converter (e.g., a current to voltage converter or an analog-to-digital converter) or a signal amplifier, for example, depending on the dynamic range of the component, its gain, and the size of the input signal.
  • analog signal 10 is then usually input into an analog-to- digital converter (AfD converter), and the A/D converter samples the analog signal 10, and outputs binary numbers via 2 n signal lines 16, where N is the number of signal lines, and output values range from 0 to 2 n -l.
  • AfD converter analog-to- digital converter
  • the digital signals output by the A/D converter 16 are input into a digital signal processor which performs signal integration. For partially saturated pixels, the signal processor processes the non-saturated signals to produce an evaluation of the pixel, e.g., data for the pixel.
  • the processing involves estimating the magnitude of any saturated signals by extrapolating from the non- saturated signals and integrates the estimated saturated signals and non-saturated signals to produce an evaluation of the pixel.
  • each pixel is usually associated with a numerical value, e.g., an integer or floating point number, representing the integrated scan signal from a region on the surface of an object (e.g., an array).
  • This evaluation may be stored in memory.
  • the time period corresponding to a pixel may be adjusted to correspond to any specified dimension representing a region of the surface of an object being scanned (e.g., such as a chemical array).
  • one feature of the invention relates to the integration of partially saturated digital signals by the signal processor.
  • the processor processes the non-saturated digital signals (e.g., will estimate the saturated digital signals for the saturated signals by extrapolating the non-saturated signals).
  • the output signal of the signal processor represents a processed (e.g., integrated) version of the input digital signals.
  • the invention provides methods for evaluating (e.g., producing a numerical evaluation of the brightness of) a pixel during scanning of a chemical array.
  • the method involves: a) producing a plurality of analog signals, e.g., signals 12 and 16, for a pixel using a multi-gain signal detection system 8, b) digitizing these analog signals using a converter (such as, for example, by using a corresponding plurality of analog-to-digital converters, e.g., 18 and 20), and, c) integrating and outputting a single integrated signal for one of the digitized signals 24 using a digital signal processor (DSP) 22.
  • DSP digital signal processor
  • the DSP 22 determines whether a signal is a non-saturated signal (e.g., signal 12) or a saturated signal (e.g., signal 16) or a partially saturated signal (not shown), and, if any non-saturated signals are detected, a single non-saturated signal is integrated and output. Accordingly, the methods find particular use in evaluating an at least partially saturated pixel, where an "at least partially saturated pixel" is a pixel represented by one or more signals that is at least partly saturated (including fully saturated), i.e., at least at a maximum amplitude.
  • multi-gain photodetection system where such a system contains at least one multi-gain system component that inputs a single signal, e.g., a light signal (in the case of a light detector, for example), a current signal (in the case of a current-to- voltage converter, for example), or a voltage signal (in the case of a voltage amplifier, for example), and outputs a plurality of analog signals (i.e., 2 or more, e.g., 3, 4, 5, 6, 7, 8, 9, 10, about 12 about 16, about 20 or more, sometimes up to about 50 or more analog signals) that are different from each other only in terms of their magnitude.
  • a light signal in the case of a light detector, for example
  • a current signal in the case of a current-to- voltage converter, for example
  • a voltage signal in the case of a voltage amplifier, for example
  • Such a system usually contains a single multi- gain system component that inputs a single analog signal (e.g., a light, current, or voltage signal) and outputs multiple analog signals that are proportional to the input signal, but at different magnitudes (except where an output signal may be saturated).
  • Exemplary gain settings may differ by a factor of about 2 or more, of about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1000 or more, about 5000 or more, about 1 x 10 4 or more, about 5 x 10 5 or more, 1 x 10 6 or more, 5 x 10 7 or more, usually up to about 1 x 10 8 or more.
  • output signals of a multi-gain system component usually differ from each other in magnitude by at least about 2-fold, by at least about 5-fold, by at least about 10-fold, by at least about 50-fold, etc.
  • a multi-gain photodetection system may contain two or more single-gain system components set at different gains.
  • a subject system may contain two or more detectors, two or more current-to-voltage converters or two or more voltage or current amplifiers at different gain settings, etc., each set at a different gain.
  • the signal input for most embodiments of the subject multi-gain photodetection systems is a single wavelength of light, corresponding to a single "channel" of light, as is commonly referred to in chemical array scanner arts.
  • “red” and “green” light emitted from fluorescent cyanine dyes may separately serve as an input light for the subject system.
  • FIG. 6 Exemplary, non-limiting, multi-gain photodetection systems useful in the subject methods are shown in Fig. 6.
  • light signal 26 enters two or more detectors 28 and 30 set at different gains, e.g., g ⁇ and gl.
  • the outputs of detectors 28 and 30 are analog current signals, and these outputs are input into current- to- voltage converters 32 and 34 to provide two analog voltage signal outputs ⁇ l and o2 that differ in magnitude according to gl and g2.
  • light signal 36 enters a multi-gain light detector 38 set at a plurality of different gains, e.g., gl and g2.
  • the outputs of detector 38 are two analog current signals, and these outputs are input into current-to- voltage converters 40 and 42 to provide two analog voltage signal outputs ol and o2 that differ in magnitude according to gl and g2.
  • light signal 44 enters single gain detector 46, and the output of this detector, an analog current, is input into a current-to-voltage converter 48 to provide an analog voltage.
  • This analog voltage is input into a multi-gain amplifier 50 set at a plurality of different gains, e.g., gl and g2, and the amplifier outputs signals ol and o2 that differ in magnitude according to gl and g2.
  • the output signal of the subject system may be any electrical signal (e.g., voltage, current, etc.). However, in many embodiments, the output signals of the system are voltage signals.
  • FIG. 5 plot signal magnitude (i) versus time (t) for a pixel, which, in this figure, is represented by a period of time that a light signal is detected.
  • saturated pixels correspond to bright areas of a scan of the surface of an array, and an illustration of a light signal that may give rise to a saturated pixel is shown as element 6 of graph 2 in Fig. 5.
  • This graph shows an exemplary light signal 6, relative to the maximum intensity of light detectable by typical prior art detector systems 4. Some of the light signal is beyond the detection limit of the typical detector system.
  • Light signal 6 is processed by multi-gain photodetection system 8, having multiple analog output signals, e.g., outputs ol and o2.
  • output ol is at a lower magnitude than output o2, and represents non-saturated signal 12, as shown in graph 10.
  • Part of the analog signal output o2 is maximal and "flat", indicating that the signal is saturated. Accordingly, output o2, as indicated in graph 14, is saturated.
  • the outputs of system 8 e.g., ol and o2 are independently sampled and digitized by analog-to-digital converters (A/D converters), e.g., elements 18 and 20.
  • A/D converters analog-to-digital converters
  • each pixel may be associated with a numerical evaluation, e.g., an integer or floating point number, representing the integrated scan signal from a region on the surface of an object being scanned (for example a chemical array). This numerical evaluation may be stored in a memory.
  • a numerical evaluation e.g., an integer or floating point number
  • Normalizing the outputs may be achieving by, for example, normalizing by the channel gain or multiplying the numerical evaluation obtained from lower gain channel by the ratio of the highest gain channel to the lower channel's gain.
  • the gain setting that was used to obtain the numerical evaluation is indicated, i.e., tagged, with the numerical evaluation in the processor output, and the normalization may happen at another time (e.g., by data extraction or data analysis software).
  • the gain used to produce a numerical evaluation may be indicated using unused bits of a binary code representing the numerical evaluation.
  • the digital signal processor 22 is programmed to integrate and output a signal corresponding to a non-saturated input signal. Accordingly, in many embodiments, the processor 22 is programmed to detect an input digital signal that is non- saturated, and select that non-saturated input signal for integration and output. In one embodiment, if a plurality (i.e., more than one) of non-saturated input signals are input into the detector, the DSP 22 integrates the non-saturated signal having the highest magnitude. In other words, if a single non-saturated input signal for a pixel is detected, it is integrated by DSP 22 to provide a numerical evaluation of the pixel.
  • the set of unsaturated input signals that has the greatest overall magnitude is integrated to provide a numerical evaluation of the pixel.
  • the non-saturated input signals are distinguished from the saturated signals by virtue of the fact that non-saturated signals are not saturated. In other words, in certain embodiment, non-saturated signals may be detected because they are not at maximal magnitude. Accordingly, in certain embodiments, for each plurality of signals input into the processor, the non-saturated signals can be identified by detecting the saturated signals.
  • the methods find use in integrating signals for pixels that are fully or partially saturated (i.e., the signal for a pixel is more than about 5%, more than about 10%, more than about 10%, more than about 20%, more than about 40%, more than about 60%, more than about 80%, more than about 90%, more than about 95%, up to 99% or 100% saturated) under scanning conditions that are typically used during array scanning, e.g., Cheung et al., Nature Genetics 1999, 21: 15-19.
  • the subject methods may be done in "real-time".
  • the single integrated signal or data for a pixel obtained using the subject methods is generally output from the processor prior to processing of the signals for the next pixel.
  • data obtained from a signal may be stored in a buffer and analyzed while accumulating data from a future pixel, e.g., the next pixel scanned.
  • the invention provides methods of evaluating a pixel (e.g., identifying and integrating a non-saturated signal for a pixel), and a system for performing the methods.
  • the system contains at least: a) multi-gain detection system (such as a multi-gain photodetection system) 8, b) one or more converters, e.g., a plurality of converters, for converting an analog signal to a digital signal (e.g., such as A/D converters 18 and 20), and c) a digital signal processor 22 that is programmed to integrate a signal and output data corresponding to a non-saturated input signal.
  • other system components may be present in the system, such as a current-to-voltage, voltage-to-current integrator, signal amplifiers, other processors, and the like.
  • the invention provides methods for identifying feature boundary pixels produced during scanning of a chemical array. These methods generally take advantage of the fact that during the scanning process, the detector of a scanner is usually in continuous or near continuous motion relative to the array being scanned. Accordingly, as the detector "passes over" (i.e., detects, while moving relative to) a boundary for a light-emitting feature, the light signal detected by the detector usually increases from background (corresponding to a non-feature area) to non-background signal (corresponding to a light-emitting feature), or vice versa.
  • a signal line (e.g., a series of individual data samples that are integrated to form a pixel) for a pixel representing the boundary for a light-emitting feature usually contains at least two parts, a first part (at either of the two ends of the signal line) corresponding to background, and a second part (at the other end of the signal line) corresponding to a feature.
  • the signal line between these two parts of the signal usually represents the feature boundary.
  • a feature boundary may span more than one pixel, e.g., two or more pixels.
  • signals for feature boundary pixels usually exhibit a marked increase or decrease in amplitude in signal over the time of the pixel.
  • a pixel signal line solely representing a non-feature area or a feature may be "flat", with no significant difference in the first and second parts of the signal line. Since the detector may scan from a non-feature area to a feature area or, alternatively, from a feature area to a non-feature area, the "background" signal part of a signal line may either be at the beginning of the signal line, or at the end of the line, respectively.
  • signal lines for feature boundary pixels may be bimodal, whereas signal lines for other pixels are not bimodal and are flat.
  • each of those pixel signals may be unimodal, having a line of best fit that is sloped along all or most of the pixel width between the beginning and end of the pixel.
  • the subject methods of determining whether a pixel signal is a signal representing a feature boundary involve evaluating changes in pixel signal amplitude over the time of the pixel.
  • Several different methods may be used to evaluate amplitude changes of a pixel signal to determine if the pixel is a feature boundary pixel.
  • these methods generally involve evaluating any difference in amplitude between a first part of a pixel signal line (i.e. a signal line representing a signal for a pixel) and a second part of that signal line, to provide an evaluation of amplitude change of the pixel signal line.
  • changes in pixel signal amplitude may be evaluated by determining a line of best fit (e.g., a linear or quadratic line of best fit normalized to the median sample) for the pixel signal.
  • a line of best fit e.g., a linear or quadratic line of best fit normalized to the median sample.
  • a pixel signal having a line of best fit having a slope larger than a certain threshold slope indicates that pixel is a feature boundary pixel.
  • a feature boundary pixel signal is a signal from pixel at the boundary of a feature.
  • Such signals generally have a significant change in amplitude over the time of the pixel.
  • a feature boundary pixel may have a signal line comprising two consecutive parts, a first part representing a signal from a feature and a second part representing a signal from a non-feature area, or a significant slope.
  • the first and second parts of a pixel signal may be in any order.
  • an area of an array corresponding to a pixel is scanned to provide a pixel signal, and any differences in amplitude of the pixel signal (e.g., between the first and second parts of the signal) are evaluated.
  • a significant difference (either an increase or a decrease) in signal amplitude indicates that the pixel signal is a feature boundary pixel signal.
  • an entire signal for a pixel is integrated to provide a single numerical evaluation of the amount of light signal emitted from an area of a chemical array. Accordingly, for a feature boundary pixel signal, background signal as well as feature signal may be integrated together, providing a single evaluation that does not accurately represent the signal for the feature represented in the pixel.
  • a pixel signal that is a feature boundary pixel signal may be detected, the pixel signal may be integrated to provide a numerical evaluation of the pixel, and the numerical evaluation may be adjusted (i.e., increased or decrease) (e.g., according to the difference in amplitude between the first and second parts of the pixel signal line or by ignoring signal that is not feature area derived) to provide a more accurate evaluation of the pixel signal.
  • the sample number at which the signal passes through the middle of the low and high range could be linked to the numerical evaluation and the background or feature signal may be defined by the samples that lie on either side of the transition sample).
  • the number of the transition sample may be recorded as an annotation associated with an output data file that may be used during feature extraction. The number could either be encoded into unused bits in the pixel data word, or saved in a separate file.
  • a pixel signal is in certain embodiments evaluated to provide a numerical evaluation, of amplitude change using the methods described above.
  • This numerical evaluation may be stored in a data file on a computer readable medium, and may be used at a later date.
  • a pixel signal may also be integrated to provide a numerical evaluation of the pixel (i.e., an evaluation of the brightness of the pixel). This numerical evaluation may also be stored in a data file on a computer readable medium, and may be used at a later date.
  • such a processor may have two outputs, one output representing the evaluation and one output representing the amplitude change evaluation of the pixel.
  • a processor may have a single output (e.g., a binary number of the like) that represents both the evaluation and the amplitude change of the pixel.
  • a processor with a 16-bit, 24-bit, 32-bit or greater than 32-bit (e.g., 64-bit or greater, etc.) word size
  • a plurality of the bits usually at least 12 bits (e.g., at least 14, 16, 20, 24, or more bits) are used to represent the pixel evaluation, whereas the remainder or a portion of the remainder of bits (usually at least 2, 4, 6, or 8 or more, usually up to about 12 or more bits) are used to represent the amplitude change.
  • Other information that could be associated with the numerical assessment includes: a) an indication of which end of the pixel (e.g., the "left” or “right” end) represents signal from background or feature (e.g., an indication of whether the pixel has a rising or falling transition), b) an indication of the percentage change of the feature, and c) an indication of how far through the pixel transition (from low to high or from high to low) is observed. For example, i.e. if a low signal has an amplitude of 1 and a high signal has an amplitude of 3 and there are 100 samples, the sample at which the signal transition from below 2 to above 2 may be recorded.
  • the processor output for a single pixel may be used by data extraction or analysis software to identify integrated signals corresponding to feature boundary pixel signals, or to adjust an evaluation of the pixel to provide a more accurate evaluation of the brightness of the pixel, as described above.
  • Graph 2 of Fig. 4 shows a representative signal line for a feature boundary pixel
  • a representative signal line for a pixel representing a feature boundary i.e., a representative signal line for a pixel representing a feature boundary
  • t time
  • i signal amplitude
  • the first part of the signal line 6 represents signal from a non-feature area (i.e., background signal)
  • the second part of the signal line 4 represents signal from a light-emitting feature.
  • such a signal line is an analog signal line that is usually the output of a detector (or a processed version thereof) that has passed over (i.e., scanned) the edge of a feature.
  • this signal line is sampled and digitized by an analog-to- digital converter, to provide a plurality of digital signals for said pixel 8. These signals are input into a digital signal processor in which certain embodiments of the invention are performed.
  • the processor evaluates amplitude change of the pixel signal 14. This evaluation may be done using straightforward or more complex methods, depending on the speed of the processor used and the desired outcome. In a simple case, amplitude change may be evaluated by determining the percent increase or decrease in the intensity of the first and second halves of a pixel signal relative to its average intensity. In other words, the first half of a pixel signal may be averaged (e.g., to produce signal line 12) the second half of a pixel signal may be averaged (e.g., to produce signal line 10) and those averages compared to the average intensity for the entire pixel signal, to provide an evaluation of the change in intensity of the pixel signal.
  • More complex methods may involve first analyzing a pixel signal line with graphing software to determine if the signal line has two proximal plateaus of different amplitude (e.g., is a signal line that can be described by a step function, e.g., a bimodal line) and, if such a signal line is detected, then comparing the signal amplitudes of the two plateaus and providing a numerical evaluation of the amplitude difference.
  • a bimodal pixel may be identified by calculating the means and standard deviations of the first half and second half of a pixel. A pixel is likely a feature boundary pixel if the difference between the means is large compared to their combined standard deviation.
  • a pulse height analyzer may be employed to generate a histogram of sample values, and identify peaks in the histogram.
  • changes in pixel signal amplitude may be evaluated by determining a line of best fit (e.g., a linear or quadratic line of best fit normalized to the median sample) for the pixel signal.
  • a line of best fit e.g., a linear or quadratic line of best fit normalized to the median sample
  • a pixel signal having a line of best fit having a slope larger than a certain threshold slope indicates that pixel is a feature boundary pixel.
  • the pixel signal may be further evaluated to determine the fit of the signal to the line of best fit. For example, the root mean square deviation from the fit could be calculated.
  • a feature boundary pixel is indicated if a pixel is a good fit to its line of best fit and its line of best fit has a significant slope (a slope greater than a pre-determined threshold slope, for example).
  • the significance of a change in amplitude of a pixel signal may vary for each scanner system used (because of the resolution of the scanner and feature size, etc.). However, the significance of a change in amplitude of a pixel signal and may experimentally determined, for each scanner system, by routine methods. For example, a scanner may be trained to recognize the shape of feature boundary pixels by scanning a feature boundary to provide a feature boundary pixel signal, and training the scanner to recognize the shape of that type of pixel signal.
  • methods of evaluating amplitude change of a pixel signal find use in a variety of applications. For example, such methods may be used to detect feature boundary pixel signals and thereby provide information about (e.g., indicate the position of) feature boundaries 16 in a scan of an biopolymeric array. Used with data extraction software, this feature boundary information may be used to extract data only from pixels representing features and not, for example, non-feature areas.
  • amplitude change of a pixel signal is evaluated to determine if the change is significant.
  • a significant change in amplitude usually indicates a feature boundary pixel signal. What constitutes a "significant” or “insignificant” change in signal intensity may easily be determined empirically.
  • pixels with a "significant" difference those with a pixel difference over a threshold, usually a pre-determined threshold, and may be pixel signals that have a greater than about two-fold (e.g., a greater than 3, 4, 5, 10, 20, 50 or 100-fold or more, usually up to about 1000-fold or more) increase or decrease in signal intensity, as compared to a pixel with no increase in signal intensity (e.g., a flat line signal).
  • a "significant" difference is one which is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 100% increase or decrease in signal.
  • a "significant" difference is one which is statistically significant (p ⁇ 0.5). In cases where the pixel signal line is flat, and depending on the exact method used for evaluation, the evaluation will be usually show that the change in signal intensity is not significant.
  • the subject methods of numerically evaluating signal amplitude change of a feature boundary pixel signal may also be used in methods of adjusting (i.e., changing or correcting) a numerical evaluation of the signal amplitude of a pixel to provide a more accurate evaluation of the brightness of a pixel 18. Again, these methods may be performed using straightforward or more complex protocols, depending on the desired result. For example, in the example set forth above, where the amplitude change may be evaluated by determining the percent increase or decrease in the intensity of the first and second parts of a pixel signal relative to its average intensity, the numerical evaluation of the pixel may be adjusted in proportion to the calculated increase or decrease.
  • the numerical evaluation may be multiplied by a factor proportional to that percentage.
  • the numerical evaluation may be increased by 500% (half of 1000%), i.e., multiplied by a factor of 5, to provide an adjusted numerical evaluation that more accurately reflects the brightness of the pixel.
  • more complex methods may be used to adjust a numerical evaluation of the signal amplitude of a pixel based on signal amplitude changes of a pixel signal, and these may generally involve standard graphing and extrapolation tools.
  • the higher plateau signal may be extrapolated to encompass the entire pixel, and the entire signal (i.e., the higher plateau signal line and the extrapolated signal line) may be integrated to provide an adjusted numerical evaluation of the pixel that more accurately represents the brightness of the pixel.
  • the samples representing the lower plateau may be ignored during integration.
  • a pixel signal may be assessed by a scanner using the methods described above to produce data, the data may be exported from the scanner and optionally stored, and a second computer may identify feature boundaries and/or adjust pixel evaluations for feature boundary pixel signals, according to the methods described above.
  • the subject methods may be done in "real-time".
  • an integrated signal i.e. an "evaluation” of the pixel
  • an evaluation of amplitude change of the signal i.e., an "amplitude change evaluation” of the pixel
  • data obtained from a signal may be stored in a buffer and analyzed while accumulating data from a future pixel, e.g., the next pixel scanned.
  • methods according to the invention involve providing (or, e.g., producing or identifying) a set of conformant signals for a pixel (i.e., a set of signals that have been filtered to remove outlier or noise signals), and integrating those conformant signals to provide an evaluation of the pixel.
  • the conformant signals can be identified in a time domain (e.g., by excluding individual samples of a pixel signal) or in a frequency domain (e.g., by Fourier transforming a pixel signal and removing frequency components that are above a threshold frequency).
  • the subject methods involve evaluating, usually by statistical methods, the plurality of digital signals to identify and exclude "outlier" signals that do not conform to a pre-determined or normal signal range of the plurality of digital signals.
  • the subject methods may involve Fourier transforming signals to identify outlier signals by their frequency. Outlier signals typically are frequency components above a threshold frequency. These methods effectively act to "filter out” undesirable portions of a pixel signal prior to integration of the pixel signal.
  • the method can be used to increase the resolution of an image by detecting pixel or sub-pixel intensity changes and adding this information to a data file for use during feature extraction.
  • This additional information could be, for instance, a percent increase or decrease in the intensity of the first and second half of a pixel (relative to its average).
  • One practical limitation to this increase in resolution would be approximately the width of the guassian beam profile convolved with a stop function. In one aspect, this increases the resolution of a scanner at least about 1-fold, at least about 2-fold or at least about 3-fold.
  • Another advantage of measuring pixel or sub-pixel spatial intensity variations is that those variations measured which are higher spatial frequency than the convolved resolution described above will be known not to be unrelated to the intended optical signal. These variations can then be flagged (annotated) and excluded from the data in some form superior to an electronic filtering of the noise (which would simply spread the noise over a wider range of acquisition time but not exclude it altogether). For example, this would be useful for the brighter signal areas of an array in a system that is not a photon shot noise limited detection system.
  • the methods may involve calculating a mean (i.e., average) signal intensity for the plurality of pixel signals, and a standard deviation of signal intensity for the signals.
  • Signals that do not conform to the normal signal range of the plurality of digital signals may be identified by determining which signals have intensities that are greater than a certain number of standard deviations, e.g., 2, 3, 4, 5, 6, 7, 8 or more, usually up to about 10 or 20 or more standard deviations, away from the mean intensity.
  • the subject methods may characterize the intensities of the pixel signals by calculating a line of best fit (that may include error bars) by regression, and by calculating which of the pixel signals do not conform to (i.e., are not described by) the line. Such methods are standard in the statistical arts. Once identified, the "outlier" signals for a pixel may be excluded or ignored when integrating the plurality of digital signals. Such methods are generally referred to as filtering in a "time" domain.
  • Graph 80 of Fig. 1 shows an analog signal line 82 for a pixel, plotted as intensity i versus intensity t in which a portion of the signal line is non-conformant 84.
  • the signal is sampled and digitized 85, to provide a plurality of digital signals for the pixel, shown in graph 86.
  • the non-conformant portion of the analog signal line is represented by digital signal 88.
  • the plurality of digital signals for the pixel are evaluated by providing a line of best fit for the signal intensities 92, including an evaluation of the range of acceptable signal intensity variation 89 and 90.
  • the non-conformant digital signal is identified because its intensity falls outside of this range, and may be ignored to provide a set of conformant digital signals for a pixel, diagrammatically illustrated in graph 98, which are integrated 99.
  • the signals intensities are averaged, and a range of acceptable signal intensity variation is provided 96, e.g., as a multiple of a standard deviation/error of the signal intensities relative to the average.
  • acceptable signal intensity variation i should be within certain limits, in this case a (e.g., the average signal intensity) +/- b (e.g., a multiple of a standard deviation/error of the signal intensities).
  • the non-conformant digital signal is identified because its intensity falls outside of this range, and may be ignored to provide a set of conformant digital signals for a pixel, diagrammatically illustrated in graph 98, which are integrated 99 to provide an evaluation of the pixel.
  • Signals may also be filtered in a "frequency" domain.
  • these methods involve Fourier transforming a signal (over a period of time) to provide the relative strength and phase of frequency components of the signal, and filtering out undesirable frequencies.
  • the maximum frequency component is related to the inverse of the minimum time between samples, and the minimum frequency component is related to the inverse of the entire length of time that is taken to acquire an entire pixel.
  • filtering using frequency domain methods generally involves Fourier transforming a signal, removing frequency components that are higher than a selected threshold frequency, and reverse Fourier transforming the data (minus the omitted frequency components).
  • the threshold frequency may be related to the maximum rate at which the signal for a pixel can change assuming that the excitation source is constant, focused to a certain size and moving at a certain speed across an array.
  • a focused laser spot of 5 microns (full width half max), w is moving across an array at a speed of 1 meter/second, v.
  • This laser passes across a perfectly sharp edge from a surface area void of fluorescent molecules to a surface area containing a significant amount of fluorescent molecules.
  • the signal increases at a frequency that is generally related to the inverse of the amount of time that it takes the entire beam (or at least half of it) to cross this perfectly sharp threshold.
  • the frequency would be the inverse of v/w (which is generally correct to within factors of order unity that are determined by the shape of the beam). In this example, it would be frequencies above the order w/v that would be excluded from the signal.
  • the threshold frequency could be either theoretically calculated or measured experimentally for the instrument in question.
  • the threshold frequency could be evaluated by scanning a sample with a sharp non-fluorescent to fluorescent transition to provide a dim to bright signal (over time, relative to the dynamic range of the scanner) that is sharp relative to the size of the spatial resolution of the scanner. If several samples are evaluated using these methods, then the frequency response of the system can be determined.
  • the frequency response of noise can also be measured in different signal intensity ranges. Using these measurements a frequency limit (or a limit for different signal intensity ranges) can be chosen that removes noise, or outlier signals, preferentially over signal in an optimal manner for that system and that signal range.
  • a pixel signal containing noise 84 is Fourier transformed, and the signal s plotted against frequency/.
  • a threshold frequency 70 (which may be arbitrarily or experimentally determined, for example) is applied to identify frequencies that are below the threshold, and those frequencies below the threshold are reverse Fourier transformed to produce a conformant signal (i.e., a signal with reduced noise).
  • This conformant signal may be integrated to produce data (e.g., such as a numerical evaluation) relating to the pixel.
  • the magnitudes of frequency components having frequencies above a threshold frequency may be reduced as a function of their frequencies.
  • the magnitude of those frequency components may each be reduced by an amount that depends on its frequency.
  • the higher the frequency the greater the reduction in magnitude.
  • the methods may provide a sliding scale of reductions to the magnitudes of a plurality of frequency components. The magnitudes may be reduced by any percentage at or between about 10% and about 100%, for example.
  • the magnitude of a frequency may be reduced by about 100% (i.e., reduced to zero), up to about 80%, up to about 50%, up to about 30% or up to about 20%, for example.
  • the amount by which a magnitude of a frequency component may be reduced may be determined by a series of pre ⁇ determined threshold frequencies. As discussed above, such threshold frequencies could be experimentally determined.
  • Both of the filtering methods described above are particularly useful in a system that is not photon-shot noise limited. Further, both of the filtering methods described above are most useful when the total number of integrated photons detected during a pixel is large compared to the total number of samples in the pixel (e.g., by a factor of about 2 or more, about5 or more or about 10 or more).
  • the subject methods may be done in "real-time".
  • the single integrated signal or data for a pixel obtained using the subject methods is generally output from the processor prior to processing of the signals for the next pixel.
  • data obtained from a signal may be stored in a buffer and analyzed while accumulating data from a future pixel, e.g., the next pixel scanned.
  • the invention also provides a variety of computer-related embodiments.
  • the methods described above may be in the form of programming for execution by a digital system processor.
  • the methods described above may be implemented through the execution of instructions stored in a computer program product in the form of programming, and the programming may be executed by a signal processor.
  • the invention provides a digital signal processor programmed to estimate, adjust and integrate saturated digital signals by extrapolating non-saturated digital signals, as discussed above.
  • the programming may be coded onto computer-readable medium, and the programming and the processor may be part of a computer-based system.
  • the invention provides a digital signal processor programmed to input multiple digital signals for a pixel, process these signals to identify a non-saturated input signal, and output data corresponding to that single non-saturated input signal, as discussed above.
  • the computer program product comprises programming coded onto computer-readable medium, and the programming and the digital system processor may be part of a computer-based system.
  • the invention provides a digital signal processor programmed to evaluate pixel signal amplitude change and/or identify feature boundary pixels, as described above.
  • the programming may be coded onto computer-readable medium, and the programming and the processor may be part of a computer based system.
  • the invention also provides feature extraction and data analysis software that uses the methods for identifying feature boundary pixels described above.
  • Such software may use the information provided by the methods described above to identify and extract data from pixels that contain only feature information, and may, in some embodiments, alter data for feature boundary pixels using the methods described above.
  • Feature extraction describes the method by which numerical data is obtained from an array.
  • feature extraction methods involve identifying a feature (usually corresponding to a probe) on a scan of a hybridized array, and measuring the amount of label (e.g., fluorescence) that is associated with the feature.
  • feature extraction methods provide a numerical figure for chosen features in each of the two or more scans of an array.
  • microarrays such as IMAGINETM by BioDiscovery (Marina Del Rey, CA) Stanford University's "ScanAlyze” Software package, Microarray Suite of Scanalytics (Fairfax, VA), "DeArray” (NIH); PATHWAYSTM by Research Genetics (Huntsville, Ala.); GEM toolsTM by Incyte Pharmaceuticals, Inc., (Palo Alto, Calif.); Imaging Research (Amersham Pharmacia Biotech, Inc., Piscataway, NJ.); the RESOL VERTM system of Rosetta (Kirkland, WA ) and the Feature Extraction Software of Agilent Technologies (Palo Alto, CA).
  • IMAGINETM by BioDiscovery (Marina Del Rey, CA) Stanford University's "ScanAlyze” Software package, Microarray Suite of Scanalytics (Fairfax, VA), "DeArray” (NIH); PATHWAYSTM by Research Genetics (Huntsville, Ala.); GEM toolsTM
  • array analysis software may combine the product of the subject methods, e.g., an indication that a certain pixel is a feature boundary pixel, with other information (i.e. topographical boundaries) in order to accurately which pixels represent features and which pixels do not represent features.
  • the above methods are coded onto a computer-readable medium in the form of "programming” or “programming products”, where the term “computer readable medium” as used herein refers to any storage or transmission medium that participates in providing instructions and/or data to a computer for execution and/or processing.
  • Examples of storage media include floppy disks, magnetic tape, CD-ROM, a hard disk drive, a ROM or integrated circuit, a magneto-optical disk, or a computer readable card such as a PCMCIA card and the like, whether or not such devices are internal or external to the computer.
  • a file containing information may be "stored” on computer readable medium, where "storing” means recording information such that it is accessible and retrievable at a later date by a computer.
  • permanent memory refers to memory that is permanent. Permanent memory is not erased by termination of the electrical supply to a computer or processor.
  • Computer hard-drive ROM i.e. ROM not used as virtual memory
  • CD-ROM compact disc-read only memory
  • floppy disk compact disc-read only memory
  • RAM Random Access Memory
  • a file in permanent memory may be editable and re-writable.
  • a "computer-based system” refers to the hardware means, software means, and data storage means used to analyze the information of the present invention.
  • the minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • input means input means
  • output means output means
  • data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.
  • to "record" data programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.
  • a "processor” references any hardware and/or software combination which will perform the functions required of it.
  • any processor herein may be a programmable digital microprocessor such as available in the form of a electronic controller, mainframe, server or personal computer (desktop or portable).
  • suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based).
  • a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.
  • the subject systems and methods find particular use in chemical, e.g., biopolymeric, array scanners.
  • a chemical array scanner that contains a system for performing the subject methods described above.
  • such scanners have a laser excitation system for emitting light from the surface of a chemical array, hardware for performing the methods described above, and, usually, a storage medium for storing data produced by scanning.
  • a subject scanner may also contain programming for executing the subject methods.
  • Such scanners typically have a laser excitation system for emitting light from the surface of an array, hardware for performing the methods described above, and, usually, a storage medium for storing data produced by scanning.
  • a scanner may also contain or communicate with a processor including programming for executing the subject methods. Since array scanners typically measure at least two, and sometimes three, four or five or more wavelengths of light from the surface of an array, a subject scanner may have a corresponding number (e.g., 2, 3, 4, 5, or more) systems for performing the subject methods. In many embodiments, a subject scanner will contain typically contain at least two such systems, corresponding to the "red” and “green” channels of light emitted in typical array experiments (Cheung et al., Nature Genetics 1999, 21: 15-19).
  • Any array scanner or device may be provided to include the above programming.
  • optical scanners of interest include those described in U.S. Patent Nos: 5,585,639; 5,760,951; 5,763,870; 6,084, 991; 6,222,664; 6,284,465; 6,329,196; 6,371,370 and 6,406,849 - the disclosures of which are herein incorporated by reference.
  • An exemplary optical scanner as may be used in the present invention is shown in FIG. 3.
  • a light system provides light from a laser 100 which passes through an electro-optic modulator (EOM) 110 with attached polarizer 120.
  • EOM electro-optic modulator
  • Each laser 100a, 100b may be of different wavelength (e.g., one providing red light and the other green) and each has its own corresponding EOM HOa, HOb and polarizer 120a, 120b.
  • the beams may be combined along a path toward a holder or caddy 200 by the use of full mirror 151 and dichroic mirror 153.
  • Controller 180 may be or include a suitably programmed processor.
  • each EOM 110 and corresponding polarizer 120 together act as a variable optical attenuator which can alter the power of an interrogating light spot exiting from the attenuator.
  • the remainder of the light from both lasers 100a, 100b is transmitted through a dichroic beam splitter 154, reflected off fully reflecting mirror 156 and focused onto an array mounted on holder 200, using optical components in beam focuser 160.
  • a subject scanner may contain more than one of 150a, and more than one of 150b, or, in alternate embodiments, 150a and 150b may be multi-gain detectors.
  • each detector 150a, 150b may be of various different types (e.g., a photo-multiplier tube (PMT), or photodiode or avalanche photodiode device (APD), such as a charge-coupled device (CCD), a charge- injection device (CID), or a complementary-metal -oxide-semiconductor detector (CMOS) device).
  • PMT photo-multiplier tube
  • APD avalanche photodiode device
  • CCD charge-coupled device
  • CID charge- injection device
  • CMOS complementary-metal -oxide-semiconductor detector
  • This detection system has a fixed focal plane.
  • a scan system causes the illuminating region in the form of a light spot from each laser 100a, 100b, and a detecting region of each detector 150a, 150b (which detecting region will form a pixel in the detected image), to be scanned across multiple regions of an array or array package mounted on holder 200.
  • the scanned regions for an array will include at least the multiple features of the array.
  • the scanning system is typically a line by line scanner, scanning the interrogating light in a line across an array when at the reading position, in a direction of arrow 166, then moving ("transitioning") the interrogating light in a direction into/out of the paper as viewed in FIG. 3 to a position at an end of a next line, and repeating the line scanning and transitioning until the entire array has been scanned.
  • a subject apparatus may scan a line multiple times before making a perpendicular transition.
  • This scanning feature is accomplished by providing a housing 164 containing mirror 158 and focuser 160, which housing 164 can be moved along a line of pixels (i.e., from left to right or the reverse as viewed in FIG. 3) by a transporter 162.
  • the second direction 192 of scanning can be provided by second transporter which may include a motor and belt (not shown) to move caddy 200 along one or more tracks.
  • the second transporter may use a same or different actuator components to accomplish coarse (a larger number of lines) movement and finer movement (a smaller number of lines).
  • directly adjacent rows are scanned.
  • "adjacent" rows may include alternating rows or rows where more than one intervening row is skipped.
  • the scanner of FIG. 3 may further include a reader (not shown) which reads an identifier from an array package.
  • a reader (not shown) which reads an identifier from an array package.
  • that reader may be a suitable bar code reader.
  • the system may also include detector 202, processor 180, and a motorized or servo- controlled adjuster 190 to move holder 200 in the direction of arrow 196 to establish correct focus for the system.
  • the detector may directly detect a partial reflection from another beamsplitter (not shown) between splitters 153 and 154.
  • autofocus system 202 may contain a position detector e.g. a quadrature position encoder, also feeding back to the CU measures the absolute position (i.e., relative to the apparatus) of the servo- controlled adjuster 190.
  • focus servo control movement 196 may occur in connection with housing 164 instead of the holder, or, if the detection system is not a fixed focal plane system, by an adjustment of laser focuser 160.
  • suitable chemical array autofocus hardware is described in pending U.S. patent application Serial No. 09/415,184 for "Apparatus And Method For Autofocus” by Dorsel, et al., filed Oct. 7, 1999, as well as European publication EP 1091229 published April 11, 2001 to the same title and inventors.
  • Controller 180 of the apparatus is connected to receive signals from detectors 150a,
  • Controller 180 also receives the signal from autofocus detector 202, and provides the control signal to EOM 110, and controls the scan system. Controller 180 contains all the necessary software to detect signals from detector 202, and regulate a motorized or servo-controlled adjuster 190 through a control loop. Controller 180 may also analyze, store, and/or output data relating to emitted signals received from detectors 150a, 150b in a known manner.
  • Controller 180 also includes a digital signal processor for performing the methods described above.
  • controller 180 includes a media reader 182 which can read a portable removable media (such as a magnetic or optical disk), and a communication module 184 which can communicate over a communication channel (such as a network, for example the internet or a telephone network) with a remote site (such as a database at which information relating to array package 30 may be stored in association with the identification 40).
  • a communication channel such as a network, for example the internet or a telephone network
  • a remote site such as a database at which information relating to array package 30 may be stored in association with the identification 40.
  • an array in a package is typically first exposed to a liquid sample.
  • This liquid sample may be placed directly on the array or introduced into a chamber through a septa in the housing of the array.
  • the array may then be washed and scanned with a liquid (such as a buffer solution) present in the chamber and in contact with the array, or it may be dried following washing.
  • a liquid such as a buffer solution
  • the identifier reader may automatically (or upon operator command) read an identifier from the array package, which may be used to e.g. retrieve information on the array layout from a database containing the identifier in association with such information.
  • a database may be a local database accessible by controller 180 (such as may be contained in a portable storage medium in drive 182.
  • the saved results from a sample exposed array, read with the methods described above, may be raw results (such as fluorescence intensity readings for each feature in one or more color channels) or may be processed results such as obtained by rejecting a reading for a feature which is below a predetermined threshold and/or forming conclusions based on the pattern read from the array (such as whether or not a particular target sequence may have been present in the sample).
  • the results of the reading may be forwarded (such as by communication of data representing the results) to a remote location if desired, and received there for further use (such as further processing).
  • the subject array scanners find use in a variety applications, where such applications are generally analyte detection applications in which the presence of a particular analyte in a given sample is detected at least qualitatively, if not quantitatively. Protocols for carrying out array assays are well known to those of skill in the art and need not be described in great detail here.
  • the sample suspected of comprising the analyte of interest is contacted with an array under conditions sufficient for the analyte to bind to its respective binding pair member that is present on the array.
  • the analyte of interest binds to the array at the site of its complementary binding member and a complex is formed on the array surface.
  • binding complex on the array surface is then detected, e.g., through use of a signal production system such as a fluorescent label present on the analyte, etc, where detection includes scanning with an optical scanner according to the present invention.
  • detection includes scanning with an optical scanner according to the present invention.
  • the presence of the analyte in the sample is then deduced from the detection of binding complexes on the substrate surface.
  • Specific analyte detection applications of interest include hybridization assays in which the nucleic acid arrays of the subject invention are employed.
  • a sample of target nucleic acids is first prepared, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of signal producing system.
  • the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected.
  • Specific hybridization assays of interest which may be practiced using the subject arrays include: gene discovery assays, differential gene expression analysis assays; nucleic acid sequencing assays, and the like.
  • arrays are arrays of polypeptide binding agents, e.g., protein arrays
  • specific applications of interest include analyte detection/proteomics applications, including those described in U.S. Patent Nos.: 4,591,570; 5,171,695; 5,436,170; 5,486,452; 5,532,128 and 6,197,599 as well as published PCT application Nos. WO 99/39210; WO 00/04832; WO 00/04389; WO 00/04390; WO 00/54046; WO 00/63701; WO 01/14425 and WO 01/40803 - the disclosures of which are herein incorporated by reference.
  • the array will typically be exposed to a sample (such as a fluorescently labeled analyte, e.g., protein containing sample) and the array then read. Reading of the array may be accomplished by illuminating the array and reading the location and intensity of resulting fluorescence at each feature of the array to detect any binding complexes on the surface of the array.
  • a sample such as a fluorescently labeled analyte, e.g., protein containing sample
  • aspects of the invention may be applicable to a variety of optical scanners including those that detect chemiluminescent or electroluminescent labels.
  • the present invention will be applicable to such scanners where powering down the scanner will result in lifetime savings, as exemplified above.
  • Certain embodiments of the invention may involve transmitting data obtained from a method described above from a first location to a remote location. Certain other embodiments of the invention may involve receiving, from a remote location, data obtained from a method described above. Kits
  • Kits for use in connection with the subject invention may also be provided.
  • Such kits usually include at least a computer readable medium including computer programming products as discussed above and, in certain kits, instructions.
  • the instructions may include installation or setup directions.
  • the instructions may include directions for use of the invention with options or combinations of options as described above.
  • the instructions include both types of information.
  • kits may serve a number of purposes.
  • the combination may be packaged and purchased as a means of upgrading an existing scanner. Alternately, the combination may be provided in connection with a new scanner in which the software is preloaded on the same. In which case, the instructions will serve as a reference manual (or a part thereof) and the computer readable medium as a backup copy to the preloaded utility.
  • the instructions are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc, including the same medium on which the program is presented.
  • the instructions are not themselves present in the kit, but means for obtaining the instructions from a remote source, e.g. via the Internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.
  • means may be provided for obtaining the subject programming from a remote source, such as by providing a web address.
  • the kit may be one in which both the instructions and software are obtained or downloaded from a remote source, as in the Internet or world wide web. Some form of access security or identification protocol may be used to limit access to those entitled to use the subject invention.
  • the means for obtaining the instructions and/or programming is generally recorded on a suitable recording medium.
  • kits may also include one or more reference arrays, e.g., two or more reference arrays for use in testing an optical scanner after software installation.

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Abstract

L’invention concerne des méthodes pour évaluer un signal de pixel produit pendant un scan de matrice chimique. Elle concerne également des systèmes et des programmes pour appliquer les méthodes objet et un scanneur de matrice pourvu de ces systèmes et programmes.
PCT/US2005/027693 2004-08-04 2005-08-03 Filtrage de signaux pendant un scan de matrice WO2006017642A2 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US10/912,463 US7818129B2 (en) 2004-08-04 2004-08-04 Detection of feature boundary pixels during array scanning
US10/912,463 2004-08-04
US10/912,661 2004-08-04
US10/912,427 US7876962B2 (en) 2004-08-04 2004-08-04 Multi-gain photodetection system for array analysis
US10/912,427 2004-08-04
US10/912,027 2004-08-04
US10/912,661 US7877212B2 (en) 2004-08-04 2004-08-04 Methods and compositions for assessing partially saturated pixel signals
US10/912,027 US7627435B2 (en) 2004-08-04 2004-08-04 Filtering of pixel signals during array scanning

Publications (2)

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WO2006017642A2 true WO2006017642A2 (fr) 2006-02-16
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091652A (en) * 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
US6329661B1 (en) * 2000-02-29 2001-12-11 The University Of Chicago Biochip scanner device
EP1271131A2 (fr) * 2001-06-29 2003-01-02 Agilent Technologies, Inc. Méthode et dispositif pour identifier la distribution non uniforme d'un signal de mesure
US20030096324A1 (en) * 2001-09-12 2003-05-22 Mikhail Matveev Methods for differential cell counts including related apparatus and software for performing same
US20030168579A1 (en) * 2002-02-28 2003-09-11 Corson John F. Signal offset for prevention of data clipping in a molecular array scanner
US20030219150A1 (en) * 2002-05-24 2003-11-27 Niles Scientific, Inc. Method, system, and computer code for finding spots defined in biological microarrays
US20040001623A1 (en) * 2002-06-19 2004-01-01 Commissariat A L'energie Atomique Image analysis process for measuring the signal on biochips
US20040033622A1 (en) * 2001-02-09 2004-02-19 Delenstarr Glenda C. Methods of identifying heterogeneous features in an image of an array

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091652A (en) * 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
US6329661B1 (en) * 2000-02-29 2001-12-11 The University Of Chicago Biochip scanner device
US20040033622A1 (en) * 2001-02-09 2004-02-19 Delenstarr Glenda C. Methods of identifying heterogeneous features in an image of an array
EP1271131A2 (fr) * 2001-06-29 2003-01-02 Agilent Technologies, Inc. Méthode et dispositif pour identifier la distribution non uniforme d'un signal de mesure
US20030096324A1 (en) * 2001-09-12 2003-05-22 Mikhail Matveev Methods for differential cell counts including related apparatus and software for performing same
US20030168579A1 (en) * 2002-02-28 2003-09-11 Corson John F. Signal offset for prevention of data clipping in a molecular array scanner
US20030219150A1 (en) * 2002-05-24 2003-11-27 Niles Scientific, Inc. Method, system, and computer code for finding spots defined in biological microarrays
US20040001623A1 (en) * 2002-06-19 2004-01-01 Commissariat A L'energie Atomique Image analysis process for measuring the signal on biochips

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