text:
Methods for programming an array scanner to scan a plurality of biopolymer arrays are provided. In the subject methods, individual scanning parameters for at least two different arrays of the plurality of arrays to be scanned are selected and input into the array scanner prior to scanning the plurality arrays. The programmed scanner then scans the plurality of arrays according to the pre-selected scanning parametersAlso provided are scanners programmed according to the subject methods, as well as computer programming that provides for programming of scanners according to the subject methods. The subject methods and compositions find use in a variety of different applications, including both genomics and proteomics applications.
1. A method of programming a biopolymer array scanner to scan a plurality of biopolymeric arrays, said method comprising:
individually selecting a scanning parameter for at least two different biopolymeric arrays of said plurality prior to using said scanner to scan said plurality of arrays.
2. The method according to
3. The method according to
4. The method according to
5. The method according to
6. The method according to
7. The method according to
8. The method according to
9. The method according to
10. The method according to
11. The method according to
12. The method according to
13. A computer-readable medium comprising program that allows an array scanner to be programmed according to the method of
14. The computer readable medium according to
15. A biopolymer array scanner comprising a computer-readable medium according to
16. A biopolymer array scanner according that allows a user to program it according to the method of
17. A method of scanning a plurality of biopolymeric arrays, said method comprising:
(a) programming a biopolymer array scanner according to the method of
(b) scanning said plurality of biopolymeric arrays arrays using said programmed scanner.
18. The method of
19. The method of
20. The method of
21. A method comprising receiving data representing said result of a scan obtained by the method of
22. A kit for use in a biopolymer array optical scanner, said kit comprising:
(a) a computer-readable medium according to
(b) instructions for operating said scanner according to said programming.
[0001] This invention relates biopolymer array scanners.
[0002] Array assays between surface bound binding agents or probes and target molecules in solution are used to detect the presence of particular biopolymers. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of binding with target molecules in solution. Such 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.
[0003] 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. Usually, 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. In most instances, the target molecules are labeled with a detectable tag such as a fluorescent tag or chemiluminescent 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. 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.
[0004] As such, 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.
[0005] The scanning equipment 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 Perkin-Elmer, Agilent,, or Axon Instruments., etc. Analysis of the data, (i.e., collection, reconstruction of image, comparison and interpretation of data) is performed with associated computer systems and commercially available software, such as Quantarray™ by Perkin-Elmer, Genepix Pro™ by Axon Instructions, Microarray Suite M by Affymetrix, as well as Feature Extraction Software and Rosetta Resolver Gene Expression Data Analysis System, both available from Agilent.
[0006] In such devices, 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 on an array substrate. The resulting fluorescence signals from the surface regions are collected either confocally (employing the same lens used to focus the laser light onto the array) or off-axis (using a separate lens positioned to one side of the lens used to focus the laser 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.
[0007] Where large numbers of arrays are to be scanned, e.g., the various arrays in a given plurality may vary from each other with respect to a number of different characteristics, including the types of probes used (e.g. polypeptide or nucleic acid), the amounts of probe deposited and the size, shape, density and position of the array of probes on the substrate, etc. Furthermore, the scanning of arrays is usually done in series and, as such, a user scanning several arrays of diverse format will typically spend a considerable amount of time removing a scanned slide from a scanner at the end of a scan, loading a new slide into the scanner, adjusting the scan parameters for the new slide, and initiating a new scan for each slide. The time taken to scan several arrays can become very significant when large numbers of diverse arrays must be scanned on a single machine.
[0008] In order to increase scanning efficiency, various high throughput scanning devices have been developed. In such devices, a plurality of arrays in a holding structure, e.g., an integrated carousel or rack, is scanned. While such devices allow one to scan a plurality of slides without having to individually load and unload each slide between scans, the utility of such devices is somewhat limited, in that each slide in the plurality is scanned according to the same scanning parameters.
[0009] Of interest would be the development of a scanning device and protocol that would allow for the high throughput scanning of a plurality of different arrays, where the scanning parameters for each array are individually selected. The present invention satisfies this need.
[0010] Relevant Literature
[0011] U.S. Pat. Nos. of interest include: 5,091,652; 5,260,578; 5,296,700; 5,324,633; 5,585,639; 5,760,951; 5,763,870; 6,084,991; 6,222,664; 6,284,465; 6,317,370 6,320,196 and 6,355,934.
[0012] Methods for programming an array scanner to scan a plurality of biopolymer arrays are provided. In the subject methods, individual scanning parameters for at least two different arrays of the plurality of arrays to be scanned are selected and input into the array scanner prior to scanning the plurality arrays. The programmed scanner then scans the plurality of arrays according to the pre-selected scanning parametersAlso provided are scanners programmed according to the subject methods, as well as computer programming that provides for programming of scanners according to the subject methods. The subject methods and compositions find use in a variety of different applications, including both genomics and proteomics applications.
[0013]FIG. 1 shows a graphical user interface for viewing scan control parameters.
[0014]FIG. 2 shows a graphical user interface for setting scan control parameters.
[0015]FIG. 3 shows a graphical user interface for setting default scan settings.
[0016]FIG. 4 shows a representation of a substrate to be scanned.
[0017]FIG. 5 shows a graphical user interface for setting a new scan region.
[0018]FIG. 6 schematically illustrates an apparatus as may be used in the present invention.
[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Still, certain elements are defined below for the sake of clarity and ease of reference.
[0020] 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), peptides (which term is used to include polypeptides 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 nonnucleotide 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. A "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. Pat. No. 5,948,902 and references cited therein (all of which are also incorporated herein by reference), regardless of the source. An "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).
[0021] An "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. In the broadest sense, 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. In many embodiments of interest, the arrays are arrays of nucleic acids, including oligonucleotides, polynucleotides, cDNAs, mRNAs, synthetic mimetics thereof, and the like. Where the arrays are arrays of nucleic acids, 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). Sometimes the arrays are arrays of polypeptides, e.g., proteins or fragments thereof. ,
[0022] Any given substrate may carry one, two, four or more or more arrays disposed on a front surface of the substrate. Depending upon the use, 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 cm2 or even less than 10 cm2. For example, features may have widths (that is, diameter, for a round spot) in the range from a 10 μm to 1.0 cm. In other embodiments each feature may have a width in the range of 1.0 μm to 1.0 mm, usually 5.0 μm to 500 μm, and more usually 10 μm to 200 μm. 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). Such 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.
[0023] Each array may cover an area of less than 100 cm2, or even less than 50 cm2, 10 cm2 or 1 cm2. In many embodiments, 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. With arrays that are read by detecting fluorescence, 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.
[0024] 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. Such methods are described in detail in, for example, the previously cited references including U.S. Pat. No. 6,242,266, U.S. Pat. No. 6,232,072, U.S. Pat. No. 6,180,351, U.S. Pat. No. 6,171,797, U.S. Pat. No. 6,323,043, U.S. patent application Serial No. 09/302,898 filed Apr. 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. Also, instead of drop deposition methods, photolithographic array fabrication methods may be used such as described in U.S. Pat. No. 5,599,695, U.S. Pat. No. 5,753,788, and U.S. Pat. No. 6,329,143. Interfeature areas need not be present particularly when the arrays are made by photolithographic methods as described in those patents.
[0025] 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. In the case of an array, 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. However, either of the "target" or "target probe" may be the one which is to be evaluated by the other (thus, either one could be an unknown mixture of polynucleotides to be evaluated by binding with the other). 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. An "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.
[0026] By "remote location," it is meant a location other than the location at which the array is present and hybridization occurs. For example, 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. As such, when one item is indicated as being "remote" from another, what is meant is that the two items are at least in different rooms or different buildings, and may be at least one mile, ten miles, or at least one hundred miles apart. "Communicating" information references transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or publicnetwork). "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.
[0027] 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. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention. The 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.
[0028] 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.
[0029] A "processor" references any hardware and/or software combination which will perform the functions required of it. For example, any processor herein may be a programmable digital microprocessor such as available in the form of a electron controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, 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). For example, 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.
[0030] A "scanner" is device for evaluating arrays. In scanners, 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. Such intensities, as a function of position, are typically referred to in the art as "pixels". Biopolymer arrays are often scanned and/or scan results are often represented at 5 or 10 micron pixel resolution. To achieve the precision required for such activity, components such as the lasers must be set and maintained with particular alignment. Scanners may be bidirectional, or unidirection, as is known in the art.
[0031] 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 Perkin-Elmer, Agilent, Packard, Axon Instruments, etc., and examples of typical scanners are described in U.S. Pat. Nos. 5;091,652; 5,760,951, 6,320,196 and 6,355,934
[0032] Methods for programming an array scanner to scan a plurality of biopolymer arrays are provided. In the subject methods, individual scanning parameters for at least two different arrays of the plurality of arrays to be scanned are selected and input into the array scanner prior to scanning the plurality arrays. The programmed scanner then scans the plurality of arrays according to the pre-selected scanning parametersAlso provided are scanners programmed according to the subject methods, as well as computer programming that provides for programming of scanners according to the subject methods. The subject methods and compositions find use in a variety of different applications, including both genomics and proteomics applications.
[0033] Before the present invention is described in such detail, however, it is to be understood that this invention is not limited to particular variations set forth and may, of course, vary. Various changes may be made to the invention described and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process act(s) or step(s), to the objective(s), spirit or scope of the present invention. All such modifications are intended to be within the scope of the claims made herein.
[0034] Methods recited herein may be carried out in any order of the recited events which is logically possible, as well as the recited order of events. Furthermore, where a range of values is provided, it is understood that every intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. Also, it is contemplated that any optional feature of the inventive variations described may be set forth and claimed independently, or in combination with any one or more of the features described herein.
[0035] The referenced items are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such material by virtue of prior invention.
[0036] Reference to a singular item, includes the possibility that there are plural of the same items present. More specifically, as used herein and in the appended claims, the singular forms "a," "an," "said" and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.
[0037] In further describing the invention in greater detail than provided in the Summary and as informed by the Background and Definitions provided above, process or program aspects of the invention are first described. This discussion is followed by a description of suitable hardware for use in the invention and potential array use.
[0038] Methodology/Programming
[0039] The subject invention provides methods for independently setting scanning parameters for the individual substrates of a plurality of substrates prior to initiation of scanning of the substrates. More specifically, the subject invention provides methods of programming a scanner to scan at least two of a plurality of different array substrates according to individually pre-selected scanning parameters. The method finds-use in pre-configuring a scanner before-initiation of scanning, and allows a user to initiate a scan run and then leave the machine unattended as it scans the plurality of substrates. In general, the methods involve independently setting parameters for individual substrates to be scanner prior to initiation of scanning of the substrates. In one embodiment, the scanning parameters are automatically imported from a database of parameters using a unique identifier, such as a barcode, associated with each individual substrate. In describing the invention, the scanning parameters are described first, followed by a description of methods for independently setting the scanning parameters for individual substrates of a plurality of substrates.
[0040] Scan Parameters
[0041] In general, scan parameters may be independently set for two or more substrates of a plurality of substrates to be scanned. Typically, once scan parameters are set for the individual substrates, the plurality of substrates is scanned according to the parameter settings for the individual substrates.
[0042] By parameter is meant a variable that can be modulated to change the way a scanner scans a particular array. The number of parameters that may be individually seletected in practicing the subject methods varies, but is typically at least 2, where the number may be as high as about 5, 10, 15 or greater, but in practice typically does not exceed about 20. A variety of different parameters may be individually preselected for one or more different arrays in a given plurality, where representative parameters include, but are not limited to:
[0043] Scan region.
[0044] The scan region is a region of the surface of a substrate that is to be scanned. In general, the scan region parameters are: 1) the size of the region to be scanned, expressed in length measurements, e.g. width A and height B, and 2) the position of the region to be scanned on the surface of the substrate, expressed in length measurements, e.g. vertical distance X and horizontal distance Y from an arbitrary position on the surface of a substrate (e.g. one corner of a substrate). Typically, the scan region corresponds to the size and position of an array or feature deposited onto the surface of a substrate, and, as such, the scan region can be set to a variety of sizes and positions. The maximum scan region is generally determined by the size of the substrate. In many embodiments where a standard microscope slide is used as a substrate, the maximum scan region is 67.2 mm�21.6 mm. In these embodiments, if the scan region parameter is not set at maximum, the parameter may be set at any size less than the maximum, such as 55 mm�21 mm or less, 50 mm�21 mm or less, 45 mm�21 mm or less or 40 mm�21 mm or less. When setting a scan region parameter, typically the distances corresponding to width A and height B and the distances X and Y are set. When the scan region parameter is set for an individual substrate, the substrate is usually scanned using those parameters.
[0045] PMT settings
[0046] Photo multiplier tube (PMT) settings for each scan channel of each slide to be scanned may be set prior to scanning. Where a PMT setting of "100" is maximum PMT and a PMT setting of "0" is off, the PMT setting for each scan channel (e.g. red or green) may be set as any integer between 1 and 100, for example, 5, 10, 20 or 50. When a PMT setting is set for each channel, an substrate is typically scanned at these PMT settings.
[0047] Scan resolution
[0048] Substrates are usually scanned at a pixel resolution of between 1 and about 100, where the integer corresponds to the pixel size, in microns. As such, a substrate scanned at pixel resolution of 5 will have pixels of 5 microns in size. When a scan resolution is set using the methods of the invention, usually at 5 or 10 microns, a substrate is typically scanned at this resolution.
[0049] Dye channels
[0050] The dye channel parameter settings determine whether information from only a first (e. g. red or Cy-5 dye), only a second (e.g. green or Cy-3 dye), or both first and second dye channels is gathered. In some embodiments of the invention, information from more than two dye channels is gathered, e.g., three, four, five or more dye channels.
[0051] Output path
[0052] This parameter allows a user to set a directory to store the data from each scan. The directory can be on a local hard disk or a storage area over the network.
[0053] Description
[0054] This parameter allows a user to associate comments with the file. The comments can become part of the output file name after the scan has been completed.
[0055] Substrate unique identifier/Barcode
[0056] In many embodiments, the unique identifier of an individual substrate may be set prior to scanning. This unique identifier, which is usually a 12 or 14 digit number derived from a barcode, allows automatic selection of several of the above parameter fields by way of a database query, where the query retrieves parameters, e.g. the scan regions for a substrate, etc., from a separate database. This separate database usually contains at least scan region parameters associated with unique substrate identifiers, but may contain all of the parameters listed above. Although any barcode system is suitable for use, barcodes of the code 128, CODABAR, code 39 and code 93 are usually used. Barcodes are usually physically associated with a substrate (e.g. stuck to or printed onto a substrate).
[0057] In the present invention, the unique identifiers may be entered manually for each slide, by typing a number or manually swiping a substrate barcode with a barcode reader. Barcodes may also be entered automatically by performing a "pre-scan" of the slide where a barcode of the slide is read by a bar-code reader built into the scanner. Usually a plurality of substrates to be scanned arranged in a rack or a carousel will be subjected to a "pre-scan" to set the unique identifier for each slide.
[0058] Scan image rotation
[0059] After scanning an individual substrate, digital representations of fluorescence patterns of individual array substrates may be rotated 90 degrees in the plane of the substrate, and flipped such that a flipped image is a mirror image of the of the original digital representation. When scan image rotation (i.e. rotation and flipping) is selected, two separate digital images will be produced, each image representing a separate channel of a scan (e.g. a "red" and a "green" channel). Scan image rotation may be selected or not selected for a each substrate, and when it is selected, the output of a scan of a substrate is automatically provided as two images, each image being a rotated and flipped representation of a channel of the scan. Selection of this parameter may also name the output of the scan. For example, in a scan of a substrate called scannamel, where the channels are red and green the outputs of a scan subjected to scan image rotation may be labeled scanname1_red and scanname1_green.
[0060] Automatic file naming
[0061] This parameter allows a user to set an output file name of a scan of an individual substrate. In general, the software will merge three fields to form an output filename. For example, Prefix1_Prefix2_scannumber, would be the output filename for a scan where "Prefix1", "Prefix2" are the set parameters of the three fields. In most embodiments, the "scannumber" entry is automatically set by scanner software to avoid duplication of file names.
[0062] Default parameter settings
[0063] In certain embodiments, default scanning parameters are independently selected for each individual substrate of a plurality of substrates. The default settings may be set for scan region, scan image rotation, PMT settings, scan resolution, dye channels, output path, description, automatic file naming, etc. as described above and below.
[0064] In many embodiments of the invention, at least one (e.g. two or three or all) of the parameters for the unique substrate identifier, scan region, PMT settings, dye channels, scan resolution, output path and description are individually set for each slide. However, in other embodiments, at least one (e.g. two or three) of parameters for the unique substrate identifier, scan region, PMT settings, dye channels, scan resolution, output path and description, scanner operator, date of scanning, error handling options, output file formats (e.g. TIFF, JPEG, etc.), post processing options (e.g. background subtraction, scan image rotation, feature extraction, etc.) and further scripts (e.g. a script that sends an e-mail) that can be run after slide scanning or processing are individually set for each slide. Default parameters may be individually set for each independent substrate.
[0065] Setting Scan Parameters for Individual Array Substrates
[0066] In general, scan parameters are typically entered into a form provided by a graphical user interface of a scanner. In many embodiments, the form provides fields into which parameters are independently set for individual substrates of a plurality of substrates. In one embodiment, a substrate for parameter setting is first chosen, and then the parameters for the substrate are set. In some embodiments scan parameters for individual substrates may be a default setting, while in other embodiments, a scan parameter for an individual substrate may be a non-default setting.
[0067] The methods of the invention may be used to independently set scanning parameters of a plurality of substrates; when the plurality of slides/substrates is usually made up of 2 or more slides that will be scanned in series on a scanner, usually in one session of scanning. As such, plurality refers to 2 or more, e.g., about 4, about 15, about 24, about 48, or more substrates. Usually the parameters for at least two of the individual substrates in a given substrate are independently set, where in many embodiments the parameters of at least 10, at least 20, at least 50, at least 75 or more, including 100 number %, of the plurality are independently set. As such, the settings for a plurality of substrates may be a mixture of default and non-default settings, where each substrate has a default or non-default setting. More than one parameter may be set for each individual slide, and parameters may be set differently between individual slides.
[0068] Having described the general principle for independently setting scan parameters for individual substrates of a plurality of substrates, an exemplary embodiment of the invention is provided below.
[0069] Non-default settings
[0070]FIG. 1 shows a graphical user interface (GUI) known as the "scan control main window" for viewing scan parameters of individual substrates. In most embodiments, independent parameter setting of individual substrates is achieved through this window. The scan control main window displays scan parameters, e.g. scan region (e.g. Scan Area 60�21.6mm), red PMT level (e.g.100), green PMT level (e.g. 100), scan resolution (e.g. 10 microns), dye channel (e.g. Red & Green), output path (e.g.) , description etc., for a individual substrates, each represented by a slot. Associated with each substrate is also a unique substrate identifier (slide ID/Barcode), which may be a number provided by a barcode, further described above and below. If a user wishes to independently set a non-default parameter for an individual slide or a group of slides a slot corresponding to the slides or group of slides may be selected e.g. by use of a mouse, and the button entitled "Edit Slot Values" is pressed. Upon pressing the "Edit Slot Values" button, an editing panel opens (displayed at the bottom of the GUI of FIG. 2), wherein the user may manually set the parameters for particular slides. In most embodiments, the user opens the editing panel, selects a slide or slides for parameter setting, modifies the scanning parameters of the slides using the windows and drop-down menus provided by the editing panel, and presses the "Set Values" button to save the settings. A user wishing to use default settings for a particular substrate may select the substrate and press the "Reset Selection" button of the editing panel to set the substrate with default settings. Further parameters, such as for automatically retrieving information from a database file, for setting the path to the database file, for scan image rotation, and for file naming for each individual substrate, etc., as described above, may also be set independently for each substrate by including the appropriate buttons, pull-down menus and windows in the editing panel of the scan control main window.
[0071] The unique substrate identifier for each individual substrate may also be set, as described below.
[0072] Unique substrate identifiers
[0073] The unique substrate identifier for an individual substrate may be a number that is manually typed or read using an external barcode reader into the editing panel of the scan control main window once a substrate has been selected.
[0074] In many embodiments, however, the individual substrate barcodes are automatically read. For automatic barcode reading, using the GUI of FIG. 2, a user selects the individual sample or samples whose barcodes are to be read using a mouse, and presses the "Read Barcode from Carousel" button. Pressing this button executes code whereby barcodes for the selected samples are read automatically using a built in scanner barcode reader. Unique substrate identifiers are automatically entered into the scan table; as shown in FIG. 2.
[0075] Default settings
[0076] To change default parameter settings, a user may select "Modify Default Settings" from the "Settings" pull-down menu of the GUI of FIG. 1 to open a "default settings editing window" (FIG. 3). Scan region, dye channel, PMT sensitivity level for both red and green channels, scan resolution, description, output path, output image split and rotation (i.e. scan image rotation, and automatic file naming parameters etc., as described above, may be set using this form.
[0077] In many embodiments, scan region options may also be automatically retrieved from a selected database (e.g. an XML or GEML database) by checking the option box shown in FIG. 3.
[0078] Setting a scan region
[0079] As discussed above, a path may be set to retrieve information from a database (e.g. an XML database) containing information regarding an individual substrate e.g. the scan region, PMT and resolution settings, the sample name and information about the probes spotted onto the surface of the sample, etc. By selecting "Modify Default Settings" from the "Settings" pull-down menu shown in FIG. I to open the GUI of FIG. 3, and pressing the "Set XML (GEML) file path" a new window opens (FIG. 4) that shows the settings for the path. A path may be chosen, or new path settings may be set by typing in a path into the "New Dir" box, or selecting by browsing using the "Browse" button. The Set XML (GEML) file path and Default Settings windows may be exited by pressing "OK". A similar path setting method may be used to set a path for individual substrates using a button on the scan control main window editing panel of FIG. 2. In this embodiment, a user would select a substrate to be scanned, check a "retrieve settings from database" box and set the path to a database in order to retrieve information about the individual substrate.
[0080] In many embodiments, when choosing settings, scan regions are selected from existing scan regions. In this embodiment, the "Attempt to retrieve from XML (GEML,) files" in the default settings editor window and the "retrieve settings from database" box of the main window editing panel should be unchecked.
[0081] New scan regions may be added to a table of scan regions by selecting "Customize Scan Region" from the "Settings" pull-down menu of the GUI of FIG. 2 to open a diagrammatic representation of a slide in a slide holder (FIG. 4). By selecting "New Region" from the "File" pull-down menu of the window of FIG. 4, a new window for adding a new scan region opens (FIG. 5), where a new scan region can be added, named and saved. In many embodiments, "X" represents the X-axis measurement for the upper left-hand corner of the scan region to the upper left hand corner of the substrate, "Y" represents the Y-axis measurement for the upper left-hand corner of the scan region to the upper left hand corner of the substrate, and the width and height measured from the upper left-hand corner of the scan region, as indicated on FIG. 5.
[0082] Once a new scan region has been created, it can be independently selected for each individual slides in the "Edit Slot Values" window of FIG. 2 by selecting a slide whose scanning parameter is to be set, selecting a scan region from pull-down menu and pressing the "Set Values" button to save the settings.
[0083] Methods of Scanning a Plurality of Slides
[0084] In general, the methods provided above for setting the parameters of individual substrates or group of substrates are repeated, as necessary, to provide settings for all individual substrates. These steps can be repeated, as necessary, to provide settings for all individual slides. Once a user has set the parameters of each of the individual substrates to be scanned, the user initiates a scan run, for example by pressing the "Scan Slot" button of FIG. 1.
[0085] Scan-parameters, are typically entered and stored in the memory of a controller. Once parameters are selected for at least two substrates of a plurality of substrates, scanning may be initiated and the individual substrates are each scanned using the parameters selected for the individual substrates.
[0086] In general, the parameters for a given individual substrate of a plurality of substrates are selected before scanning of the plurality of substrates is initiated. Once scanning has been initiated, the first substrate is scanned at the parameters selected for the first substrate. After completion of scanning of the first substrate, the second slide is automatically scanned at the parameters selected for the second substrate, and so on, until the plurality of substrates has been scanned. As such, the methods described above are particularly useful in automatically scanning a plurality of substrates (e.g. biopolymeric array substrates or slides) where a user can load a machine with several substrates, set the scanning parameters for each individual substrate, initiate the scan of the plurality of substrates, and leave the scanner unattended until all the substrates have been scanned.
[0087] In general the above methods are useful in scanning a plurality of samples, where the plurality of samples have been loaded into a scanner using a rack or carousel. Scanners that can be loaded with 2 or more, 4 or more, 8 or more, 15 or more, 24 or more or 48 or more substrates are of particular suitability for use with the above methods. One such scanner suitable is described below.
[0088] Programming according to the present invention, i.e., programming that allows one to preprogram a scanner as described above, can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture that includes a recording of the present programming/algorithms for carrying out the above described methodology.
[0089] In certain embodiments, the programming is further characterized in that it provides a user interface, where the user interface presents to a user the option of selecting among one or more different, including multiple different, rules for individually pre-selecting scanning parameters, as described above.
[0090] Optical Scanners
[0091] Also provided by the subject invention are biopolymer array optical scanners that are programmed as described above. Any biopolymer optical scanner or device may be provided to include the above programming. Representative optical scanners of interest include those described in U.S. Pat. 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. 6.
[0092] Referring now to FIG. 6, an apparatus of the present invention (which may be generally referenced as an array "scanner") is illustrated. A light system provides light from a laser 100 which passes through an electro-optic modulator (EOM) 110 with attached polarizer 120. Each laser 100 a, 1 00 b may be of different wavelength (for example, one providing red light and the other green) and each has its own corresponding EOM 110 a, 110 b and polarizer 120 a, 120 b. The beams may be combined along a path toward a holder 200 by the use of full mirror 151 and dichroic mirror 153 . A control signal in the form of a variable voltage applied. to each corresponding EOM 110 a, 110 b by the, controller (CU) 180, changes the polarization of the exiting light which is thus more or less attenuated by the corresponding polarizer 120 a, 120 b. Controller 180 may be or include a suitably programmed processor. Thus, 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 in a manner, and for purposes, such as described in U.S. Pat. No. 6,406,849. The remainder of the light from both lasers 100 a, 100 b is transmitted through a dichroic beam splitter 154, reflected off fully reflecting mirror 156 and focused onto either an array 12 of an array package 30 mounted on holder 200, or a calibration member 230, whichever is at a reading position, using optical components in beam focuser 160. Light emitted, in particular fluorescence, at two different wavelengths (for example, green and red light) from features 16, in response to the interrogating light, is imaged using the same optics in focuser/scanner 160, and is reflected off mirrors 156 and 154. The two different wavelengths are separated by a further dichroic mirror 158 and are passed to respective detectors 150 a and 150 b. More optical components (not shown) may be used between the dichroic and each detector 150 a, 150 b (such as lenses, pinholes, filters, fibers etc.) and each detector 150 a, 150 b may be of various different types (e.g. a photo-multiplier tube (PMT) or a CCD or an avalanche photodiode (APD)). All of the optical components through which light emitted from an array 12 or calibration member 230 in response to the illuminating laser light, passes to detectors 150 a, 150 b, together with those detectors, form a detection system. This detection system has a fixed focal plane.
[0093] A scan system causes the illuminating region in the form of a light spot from each laser 100 a, 100 b, and a detecting region of each detector 150 a, 150 b (which detecting region will form a pixel in the detected image), to be scanned across multiple regions of an array package 30 mounted-on holder 200. The scanned regions for an array 12 will include at least the multiple features 16 of the array. In particular the scanning system is typically a line by line scanner, scanning the interrogating. light in a line across an array 12 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. 6 to a position at an end of a next line, and repeating the line scanning and transitioning until the entire array 12 has been scanned. This can be accomplished by providing a housing 164 containing mirror 158 and focuser 160, which housing 164 can be moved along a line of pixels (that is, from left to right or the reverse as viewed in FIG. 6) by a transporter 162. The second direction 192 of scanning (line transitioning) can be provided by second transporter which may include a motor and belt (not shown) to move holder 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). The reader of FIG. 6 may further include a reader (not shown) which reads an identifier from an array package 30. When identifier 40 is in the form of a bar code, that reader may be a suitable bar code reader. The reader of FIG X may also include a multi-substrate carousel or rack (not shown) which may be loaded with plurality of substrate and placed in the reader. Reader contains means for automatic means for removing a substrate from holder 200, removing a substrate from the carousel, placing the substrate removed from the carousel onto the holder 200. This means allows the automatic scanning of a plurality of slides loaded into the carousel or rack.
[0094] An autofocus detector 170 is also provided to sense any offset between different regions of array 12 when in the reading position, and a determined position of the focal plane of the detection system. An autofocus system includes detector 170, processor 180, and a motorized adjuster to move holder in the direction of arrow 196. A suitable chemical array autofocus system 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. incorporated herein by reference, as well as European publication EP 1091229 published Apr. 11, 2001 under the same title and inventors.
[0095] Controller 180 of the apparatus is connected to receive signals from detectors 150 a, 150 b (these different signals being different "channels"), namely a signal which results at each of the multiple detected wavelengths from emitted light for each scanned region of array 12 when at the reading position mounted in holder 200. Controller 180 also receives the signal from autofocus offset detector 170, and provides the control signal to EOM 110, and controls the scan system. Parameter settings entered using the methods of the are stored in Controller 180, and used by Controller 180 to perform a scan run. Controller 180 may also analyze, store, and/or output data relating to emitted signals received from detectors 150 a, 150 b in a known manner. Controller 180 may include a computer in the form of a programmable digital processor, and include 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). Controller 180 is suitably programmed to execute all of the steps required by it during operation of the apparatus, as discussed further below. Alternatively, controller 180 may be any hardware or hardware/software combination which can execute those steps.
[0096] In one mode of operation, the array is typically first exposed to a liquid sample (for example, placed directly on the array substrate). The array may then be washed and scanned with a liquid (such as a buffer solution) present on the array, e.g., in an array chamber and in contact with the array, or it may be dried following washing. Following a given array being mounted in the apparatus, the identifier reader may automatically (or upon operator command) read the array identifier (such as a bar code on the arrays substrate or housing), and use this to retrieve information on the array layout (including characteristics of the array features, such as size, location, and composition). Such information may be retrieved directly from the contents of the read identifier when the read identifier contains such information. Alternatively, the read identifier may be used to retrieve such information from a database containing the identifier in association with such information. Such a database may be a local database accessible by controller 180 (such as may be contained in a portable storage medium in drive 182 which is associated with the array, such as by physical association in a same package with the array when received by the user, or by a suitable identification), or may be a remote database accessible by controller 180 through communication module 184 and a suitable communication channel (not shown).
[0097] Utility
[0098] The subject biopolymer array scanners programmed according to the subject invention find use in a variety applications, where such applications are generally analyte detection applications in which the presence of a particular analyte in a plurality of samples 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. Generally, 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. Thus, if the analyte of interest is present in the sample, it binds to the array at the site of its complementary binding member and a complex is formed on the array surface. The presence of this 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. The presence of the analyte in the sample is then deduced from the detection of binding complexes on the substrate surface.
[0099] Specific analyte detection applications of interest include hybridization assays in which the nucleic acid arrays of the subject invention are employed. In these assays, 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. Following sample preparation, 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. References describing methods of using arrays in various applications include U.S. Pat. Nos.: 5,143,854; 5,324,644; 5,288,644; 5,324,633; 32,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992—the disclosures of which are herein incorporated by reference.
[0100] Where the 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. Pat. Nos. 4,591,570; 5,171,695; 5,436,170; 5,486,452; 00/0832,128 and 6,197,599 as well as published PCT application Nos. WO 99/39210; WO 01/4832; WO 00/04389; WO 00/04390; WO 00/54046; WO 00/63701; WO 01/14425 and 01/40803—the disclosures of which are herein incorporated by reference.
[0101] In using an array in connection with a programmed scanner according to the present invention, 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.
[0102] It is further noted that 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.
[0103] In any case, results from reading an array 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 (processed or not) may be forwarded (such as by communication) to a remote location if desired, and received there for further use (such as further processing). Stated otherwise, in certain variations, 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.
[0104] Kits
[0105] Kits for use in connection with the subject invention may also be provided. Such kits preferably include at least a computer readable medium including programming as discussed above and 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. In certain embodiments; the instructions include both types of information.
[0106] Providing the software and instructions as a kit 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.
[0107] The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, 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. In other embodiments, 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.
[0108] In yet other embodiments, 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. Conversely, means may be provided for obtaining the subject programming from a remote source, such as by providing a web address. Still further, 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. As with the instructions, the means for obtaining the instructions and/or programming is generally recorded on a suitable recording medium.
[0109] It is evident from the above discussion that the subject invention provides an important breakthrough in the ability to scan large numbers of arrays automatically. Specifically, the subject invention allows one to scan a large number of arrays automatically in a high througphut device, where each array is scanned according to individually preselected scanning parameters, so that the results obtained are identical to results that would be obtained if the arrays had not been scanned in a high throughput fashion, but instead scanned individually. Accordingly, the subject invention represents a significant contribution to the art.
[0110] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
[0111] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
Referenced by
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By Catholic Online (NEWS CONSORTIUM)
7/9/2013 (1 year ago)
Catholic Online (www.catholic.org)
Libertarian hero and NSA whistleblower Edward Snowden apparently told Der Spiegel that the United States worked with Israel to develop an ultra-powerful computer virus that successfully delayed the Iranian nuclear program. He has also been granted Asylum in Venezuela, but it remains unclear how he might travel there from Moscow.
By Catholic Online (NEWS CONSORTIUM)
Catholic Online (www.catholic.org)
7/9/2013 (1 year ago)
Published in Europe
Keywords: Snowden, Stuxnet, Moscow, Venezuela, asylum, travel, extradition, virus
LOS ANGELES, CA (Catholic Online) - According to Snowden in his interview with the German newspaper, Der Spiegel, the United States cooperated with Israel in developing the Stuxnet virus, which was successful in infiltrating Iranian nuclear facilities. The virus, or more accurately, computer worm, was designed to affect only very specific computer systems and equipment, allowing targeted harm to be done primarily to centrifuges used to enrich uranium to weapons grade.
The Stuxnet worm has long been suspected of being part of a larger joint-U.S.-Israeli operation dubbed "Olympic Games," so Snowden's revelation at this time only provides additional credibility to those claims.
Snowden said the operation was coordinated between the NSA and Israel by the Foreign Affairs Directorate, an office that manages intelligence relationships between the U.S. and other nations.
Meanwhile, the greater question is what Snowden intends to do next. Venezuela has granted asylum to the hunted Edward Snowden, but there appears no simple way for him to reach Caracas. There are no direct flights between Moscow and the Venezuelan capital.
Other countries have been asked by the United States to detain Snowden, should he attempt to pass through one of their airports. If arrested, Snowden would face charges of espionage in a U.S. court and could face decades behind bars.
At this time, Snowden will have to make at least one layover and must find a country that does not enjoy an extradition treaty with the United States. Thus far, no country is readily known. It remains unlikely that he will be able to find one and is traveling without a passport.
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By Catholic Online (NEWS CONSORTIUM)
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subset: commoncrawl


text:
Archive for the 'Press Releases' Category
September 12th, 2012 at 11:04 am
espnW: Women + Sports Summit Brings Together Top Female Athletes & Innovators Oct. 1-3
Attendees include Aly Raisman, Adam Silver, Gabrielle Reece, Julie Foudy and Sheryl Swoopes; Select Panels Available for Viewing Via Livestream[Read-More]
September 12th, 2012 at 9:38 am
ESPN3 Presents Exclusive Live Coverage of the World Twenty20 Championship in the U.S.
ESPNcricinfo to provide the latest news and highlights on multiple platforms worldwide[Read-More]
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NBC Sports Network has Kansas City hosting Houston, Fri. at 8:30 p.m. ET[Read-More]
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SEC "Storied" film about Sylvester Croom, narrated by Terrence Howard, will focus on journey of first African American head coach in the[Read-More]
September 12th, 2012 at 8:07 am
University of Oklahoma and FOX Sports Announce Long-Term Multimedia Partnership
At Least 1,000 Hours of Sooner Programming to be Distributed Annually on FOX Sports Networks in Oklahoma, Texas, Arkansas, and Louisiana[Read-More]
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and up 175% from 5.9 million viewers in 2010 (Nadal def. Djokovic) according to estimates by Nielsen.[Read-More]
September 11th, 2012 at 1:44 pm
NBC Sports Network to Televise 24 Collegge Hockey Games in 2012-13
ECAC, Hockey East, Denver, Notre Dame & Nebraska-Omaha Comprise Schedule[Read-More]


text:
After President Obama's Cairo speech, one of the more bizarre memes to emerge from the Magical Dungeon of Nonsensical Anti-Obama Talking Points was the notion that Obama's words were somehow harshly critical of Israel.
The most popular criticism: Obama's speech threw Israel "under the bus." Michael Goldfarb of the Weekly Standard said it. Former Bush One Deputy Undersecretary of Defense Jed Babbin said it. Best-selling professional Muslim-hater Robert Spencer said it, too.
Google — or, if you prefer, Bing — the phrase "Obama threw Israel under the bus" and you'll find countless examples. By countless, I mean there were a lot, but I didn't count them.
Rush Limbaugh didn't use "under the bus." Instead, he said Obama's speech "threw Israel over the cliff."
I'm not sure if getting thrown over a cliff is supposed to be better or worse than getting thrown under a bus. I suppose it depends on what's at the bottom of the cliff. If there's a giant trampoline or a nude beach with exceptionally bouncy sand at the bottom, then maybe the cliff ain't so bad.
If, however, there are dragons at the bottom of the cliff, or a freaky modern art installation consisting of thousands of upward-facing pitchforks, then I'm taking the under-the-bus option.
Charles Krauthammer of the Washington Post skipped the bus/cliff metaphors. Instead, the ol' kabbage krusher called Obama "dishonorable" because, he says, the speech bullied Israel.
Bullied how? Let's go to the transcript.
"America's strong bonds with Israel are well-known," Obama said. "This bond is unbreakable. It is based upon cultural and historical ties, and the recognition that the aspiration for a Jewish homeland is rooted in a tragic history that cannot be denied."
Nothing offensive about that. It's not like he blurted out "Opposite Day!" right afterward.
In fact, he followed it up by condemning the horror of Nazism and conflating Israel's modern-day enemies Hamas, and Iran's theocratic government with Nazis. Again, nothing about that would offend even the most easily offended friend o' Israel.
Some were offended by this:
"On the other hand, it is also undeniable that the Palestinian people — Muslims and Christians — have suffered in pursuit of a homeland. For more than 60 years they've endured the pain of dislocation," Obama said. "Many wait in refugee camps in the West Bank, Gaza, and neighboring lands for a life of peace and security that they have never been able to lead. They endure the daily humiliations — large and small — that come with occupation."
Critics of the speech said the above passage equated Palestinian suffering with the Holocaust. It didn't. It doesn't.
It's a simple acknowledgement of the pain and misery Palestinians have endured since Israel's creation. The Holocaust is the greatest act of evil ever committed. But the fact of the Holocaust doesn't cancel, mitigate or soothe the suffering of Palestinians. Ask a Palestinian if you don't believe me. Acknowledging the suffering of Jews and Arabs during the same speech isn't anti-Israeli. It's pro-human.
Obama followed that with bland pronouncements in support of a two-state solution — an Israel and Palestine side-by-side, in peace. You know, like ebony and ivory, except they're both pretty much the same shade of beige.
There's nothing anti-Israeli about that. And even though it pissed off the Krauthammers of the world, there was nothing anti-Israeli about Obama's next statement about Israel, either:
"The United States does not accept the legitimacy of continued Israeli settlements. This construction violates previous agreements and undermines efforts to achieve peace. It is time for these settlements to stop."
The settlements Obama demands Israel stop growing aren't in Israel. Is it anti-Israeli to demand Israel stop building or expanding settlements — illegal settlements — on parts of the occupied West Bank? Only if your definition of "pro-Israeli" is "blind support for the maximalist demands of Israel's right-wing political parties."
Interestingly, you know who doesn't think it's anti-Israeli to halt the growth of settlements in the West Bank? Israelis. A majority polled after Obama's speech said they agree with a settlement freeze.


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An altercation over a girl may have led to the shooting of a teenager Tuesday night in Hinesville.
Dec. 15: • Cruelty to an animal, Timber Ridge Trail
Between rising utility costs and shrinking incomes, more people are exploring alternate means of heating their homes. However, when folks misuse electric appliances or are careless around open flames they risk starting fires. Local fire investigators offered these tips for keeping safe this Christmas and all winter long.
A vacationing Long County man lost his home and his family's Christmas presents when his residence on the 200 block of Archer Road, just off Elam Road, was destroyed by fire early Sunday morning.
A Ludowici police officer has been cleared of any wrongdoing regarding his actions in the recent apprehension and nonfatal shooting of a burglary suspect.
The driver involved in a fatal accident on Oct. 19 has been arrested without incident and charged with first degree vehicular homicide.
Two men were arrested this week for an Oct. 30 home invasion and armed robbery in Ludowici; one of them also is a suspect in a Dec. 7 break-in. Two other suspects in the Oct. 30 incident are still on the loose and one is considered armed and dangerous.
Fort Stewart authorities released the name of a 3rd Infantry Division soldier who was stabbed to death Dec. 8.
A Fort Stewart military police officer was arrested in a sex sting operation Friday for allegedly soliciting underage sex online.
Andrea Renee Wilson, a co-defendant in the murder of her cousin, Prince C. Davis, was sentenced to life without the possibility of parole during a hearing Monday at the Liberty Jail.
A 24-year-old man was shot and killed Tuesday night at Pine Crest Village in Long County, according to Long County Sheriff's Department Chief Investigator Nickey Anderson.
A Hinesville gun shop owner who pleaded guilty to dealing in stolen military equipment was sentenced earlier this week.
A man was taken to a hospital after he was pulled out of a mobile home fire at 82 Pipkin Road Tuesday morning. It was one of two fires called in to Hinesville Fire Department about the same time.
Charges were filed Wednesday against the 20-year-old victim of the recent shooting at the Plantation Apartments on Casey Drive in Richmond Hill.
A construction worker helping build the new middle school in South Bryan fell to his death on Thursday morning.
A vehicle struck a home in the 700 block of Talmadge Road in Walthourville on Wednesday night, authorities said.
A woman stabbed her husband in the neck after he attacked her in Hinesville on Wednesday evening, police said.
Ludowici police officers will soon have a new piece of equipment - body-worn cameras - when they are on duty.
Recent reports filed by Liberty County Sheriff's deputies include:
© 2015 Hinesville Publishing - All Rights Reserved
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Celebrity Central / Top 25 Celebs
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Rolo – SB Warm Up Sessions
North London's Rolo warms up a session with some truth.
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Can you suggest a library I could use to instrument my .NET desktop applications to collect the UI usage information from users - how and when they move the mouse, navigate menus, click buttons, work with combo and list boxes, etc.
So after each user session or some period of time I could have a snapshot information about all user actions with my application.
If that library would come with the analysis module which could help to analyse the usage data, build a heat map and provide some recomendations on how to change the UI to make it better. That is - the "UI profiler". Or at the very least if it provide a "usage data player", so I could re-play the user session using the application on my local PC or in some schematic way.
The library itself should be a non-intrusive to allow "injecting" the UI stat collection code without changing the existing application (.NET) much.
If you could recomend a free/opensource library which does that - it would be great, but I don't mind to buy a commercial one if it does what I need.


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LARGEST - Wooden Fountain of Europe - Valendas, GR, Switzerland
in Superlatives
The old village fountain in the center of Valendas is the largest wooden fountain of Europe.
posted by: fi67
location: Graubuenden (GR), Switzerland
date approved: 12/10/2014
last visited: never
ONLY--Byzantine-Roman church in Brussels, Belgium
This old church is St. Mary's Church, the only Byzantine-Roman church in all of Brussels, Belgium and is a focal point in downtown Brussels.
posted by: Queens Blessing
location: Liège, Belgium
date approved: 12/6/2014
HIGHEST - Building in the Ruhr area, Essen, Germany
The RWE Tower in Essen is the highest building in the Ruhr area
posted by: g300td
location: Nordrhein-Westfalen, Germany
date approved: 12/4/2014
last visited: 2/17/2013
Only - Mainland Royal Albatross Colony in the World - Dunedin, New Zealand
The Royal Albatross Centre provides wildlife and cultural tours of Taiaroa Head where you can visit the world's only mainland breeding colony of Royal Albatross and see the Little Blue Penguin and the Yellow-Eyed Penguin colonies.
posted by: denben
location: South Island, New Zealand
date approved: 12/3/2014
LARGEST - Cinema Hall in Germany, Essen, Germany
Lichtburg is the cinema with the largest cinema hall in Germany
last visited: 6/21/2015
LARGEST - collection of insulators and surge arrestors in Europe - Dvorište, okres Pelhrimov, CZ
The gigantic collection, enrolled at the Ministry of Culture as a preserved technical collection, is the largest collection of insulators and surge arrestors in Europe.
posted by: Noe1
location: Kraj Vysocina, Czech Republic
OLDEST rafters in Germany
Die evangelische Martinskirche in Dautphe hat das wohl älteste Dachgestühl Deutschlands | The roof of the Protestant Martin Church in Dautphe probably has the oldest rafters in Germany
posted by: Eibacher Kuckuck
location: Hessen, Germany
date approved: 11/23/2014
BIGGEST -- Rock Crystal in the world - Stein, Germany, BY
At the entrance lobby of the mineral bath 'Kristall Palm Beach' in Stein bei Nürnberg, Germany, Bayern is the biggest rock chrystal in the world on a permanent display.
posted by: Tante.Hossi
location: Bayern, Germany
date approved: 11/20/2014
last visited: 4/20/2015
LARGEST - Solar Bridge - Blackfriars Railway Bridge, London, UK
This bridge, Blackfriars Railway Bridge, connects Blackfriars station, on the north side of the River Thames, with the south side of the Thames. The bridge incorporates the station's platforms and is covered with solar panels being used in the roof.
posted by: Master Mariner
location: London, United Kingdom
date approved: 11/19/2014
last visited: 5/24/2015
FIRST - Oil strike in State of Oklahoma, USA.
The oil derrick stands 194 feet high and is the historic site of the first oil strike on June 25, 1901 in the State of Oklahoma, the first of many that made Tulsa the Oil Capital of the World.
posted by: veritas vita
location: Oklahoma
date approved: 11/17/2014
last visited: 5/15/2014
OLDEST - of the Seven Wonders of the Ancient World
Great Pyramid of Khufu, Giza, Egypt
location: Egypt
date approved: 11/15/2014
LOWEST - Geocache on Earth
Near the shore of the Dead Sea in Jordan
location: Jordan
LONGEST - Colonnade in the Roman World
The Great Colonnade at Apamea is situated in northern Syria.
location: Syria
date approved: 11/11/2014
MIGHTIEST - Waterfall in Europe
Dettifoss is situated in the Northeast of Iceland.
location: Iceland
OLDEST - Monumental structure made of stone
Pyramid of Djoser is situated in Saqqara, Egypt.
last visited: 11/7/2014
OLDEST - Movie theatre in Nordrhein-Westfalen
Film Studio Glückauf is the oldest moovie-theatre not only in Essen but also in Nordrhein-Westfalen.
OLDEST- Solar observatory in the world
This is the oldest known Solar observatory in the world and it is situated in Goseck, Sachsen-Anhalt, Germany.
location: Sachsen-Anhalt, Germany
LARGEST - Gothic church in Northern Europe , Köln, Germany
Germany's most visited landmark
last visited: 5/13/2015
LONGEST - railway bridge in Czech Republic - Karlín, Praha, CZ
Negrelliho viadukt - the longest railway bridge in the Czech Republic and one of the longest ever built in Europe in the 19th century.
location: Hlavni mesto Praha, Czech Republic
date approved: 11/8/2014
last visited: 3/14/2015
OLDEST - Theater in a US Prison - Deer Lodge, MT
In 1919 a first in US Prison history occurred within the Montana State Prison. A theatre was built with funds ($10,000) donated by Warden Conley's personal friend William A. Clark, one of the Butte, Montana Copper Kings.
posted by: BK-Hunters
location: Montana
date approved: 11/3/2014
World's Largest Putter - Bow Island, AB
There are lots of "World's Largest" things to be found on the highways and byways of Alberta, and here's one of them, a putter.
location: British Columbia, Canada
date approved: 11/1/2014
OLDEST church in Luxembourg
In 1355 a natual cave was converted into a place to hold worship. Today, the church is the oldest church in Luxembourg.
location: Luxembourg
OLDEST Canadian Geocache - East River, Nova Scotia
OLDEST Canadian Geocache located near the Highway 103, East River, Nova Scotia, Canada
posted by: oiseau_ca
location: Nova Scotia, Canada
last visited: 1/6/2015
Youngest Member Corps of Discovery - Palmyra, MO
Later became a lawyer, and died in Palmyra while pursuing his law practice
posted by: YoSam.
location: Missouri
date approved: 10/31/2014
Oldest - Bank in the Texas Panhandle ~ Panhandle, TX
Even the VW looks old
location: Texas
date approved: 10/30/2014
last visited: 11/1/2014
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Posted by Josh Katzowitz
UPDATED 7:06 p.m. ET: According to NFL.com, Campbell will miss the rest of the season with his dislocated shoulder.
Raiders quarterback Jason Campbell has been knocked out of the Oakland-Cleveland game with about 4 minutes left in the second quarter, and according to the San Francisco Chronicle, he's dislocated his shoulder. After gaining seven yards on a run, Cleveland's Scott Fujita tackled him, and Campbell stayed on the turf until Oakland trainers could reach him.
As he slowly walked off the field, Campbell -- who was 6 of 9 for 52 yards and had helped the Raiders to a 14-7 lead against the Browns -- Campbell, in obvious pain, held his arm close to abdomen. Kyle Boller is in to replace Campbell, and on his first play in the game, a fourth and 1 from the Raiders 34, the team went for it and Boller gained the first down on a sneak.
If Boller were to go down, Terrelle Pryor -- who is not on the active roster -- would not play. Instead, the Raiders emergency third quarterback is punter Shane Lechler.
For more NFL news, rumors and analysis, follow @EyeOnNFL on Twitter and subscribe to our RSS Feed.
Blog Entry
Report: Campbell's dislocated shoulder ends year
Posted on: October 16, 2011 5:30 pm
Edited on: October 16, 2011 7:09 pm
Category: NFL
Since: Aug 7, 2008
Posted on: October 17, 2011 12:13 am
Report: Campbell's dislocated shoulder ends year
If the Raiders want to save this season then the best choice out there is picking up David Garrard but he will cost the Raiders some money for just this season but no matter what the loss of Jason Campbell will defiantly hurt them and a winning record to because I doubt that Garrard has anything left in the tank for him to take the Raiders to the playoffs. It looks like the Raiders will miss the playoffs once again like usually.
Since: Sep 14, 2006
Posted on: October 17, 2011 12:07 am
Report: Campbell's dislocated shoulder ends year
Aaron Pryor was a boxer. LOL
Since: May 16, 2008
Posted on: October 16, 2011 11:43 pm
Report: Campbell's dislocated shoulder ends year
Who the hell is Arron Pryor?
Since: Dec 15, 2006
Posted on: October 16, 2011 11:25 pm
Report: Campbell's dislocated shoulder ends year
Now that Campbell"s year is over the Raiders need a starting QB and I know who they should turn to and sign. David Garrard the ex Jacksonville Jaguar with 5 years of starting experience and a winning record against the best of the NFL. If not Garrard who???? I don't believe in Kyle Boller and Arron Pryor will get killed with his unexperience. If Garrard isn't your man you tell me who????
Since: Mar 12, 2007
Posted on: October 16, 2011 11:24 pm
Report: Campbell's dislocated shoulder ends year
I don't think Cincy will just give Palmer away. Plus the Raiders have very few draft picks next year and Cincy will want a lot in return. You can have Orton for a conditional draft pick and take some of his salary responsibility. Change of scenery might do him well and with Oakland's strong running game will keep him from making mistakes. Just my opinion.
Since: Dec 30, 2007
Posted on: October 16, 2011 11:10 pm
Report: Campbell's dislocated shoulder ends year
Unfortunate circumstances for the Raider Nation and all, but I wouldn't get down and out about the whole issue. The Raiders are a team on the rise and I believe the system that is in place wouldn't make or break the Quarterback over there. Since the Raiders love to run the ball, that places less responsibility on our QB. With that said, I am worried about Kyle Boller because he is turnover prone. And I know for a fact if he is the starter, then opposing defenses will gladly stack the box on us from here on out and dare Kyle Boller to throw. That isn't good at all....Not for him and not for the Raiders. Hue Jackson better evaluate and evaluate his QB's closely as well as all free agents. Carson Palmer would go to Oakland in a heartbeat. Question is does he still have it or not? A definite improvement over Boller no doubt. But the Bengal's Owner will be hardcore and I doubt we can grab Carson. Some people have been saying David Garrard. That wouldn't be a bad pick up at all. I think David Garrard's style of play would fit for us. However, will he be able to handle himself out there. He hasn't played in a while, plus he has to get a good understanding of our offense quickly. For that reason alone, I believe Hue Jackson might get brave and end up starting Terelle Pryor. Al Davis would have wanted Pryor to play anyways. And Al would tell Hue "you don't adjust, you dominate" which means Hue better get Pryor on the same page with him and make Pryor understand quickly. What better way to do it by sticking Pryor out there as soon as possible? I can't envision Hue Jackson making Kyle Boller our full time starter. I just don't see it...Not sure if the Raider brass wants to consider free agency or a trade. But my bet is on Hue starting Terelle Pryor. And if it doesn't happen now, it will happen real soon. Do I think Terelle Pryor deserves to start right now? No I don't. But I believe Hue will get him out there and try to make sure the kid is ready to run our offense and ready to handle NFL defenses. Hue will try to get Pryor running our offense as soon as possible. With Jason Campbell out for the year now, it might happen now. We shall see. But our season isn't over yet, and I think Hue will figure it out. One way or the other, we win the AFC West. Have faith Raider brethren.
Since: Oct 14, 2006
Posted on: October 16, 2011 11:09 pm
Report: Campbell's dislocated shoulder ends year
Is David Garrard still umemployed? If he is I think he should be our first phone call.
Since: Oct 16, 2011
Posted on: October 16, 2011 11:08 pm
Report: Campbell's dislocated shoulder ends year
I agree clock management needs to get better. As impressive as the 7-minute drive was in Q4 Boller was constantly snapping the ball with +10 left on the play clock. They could have knocked off another 60-90 seconds which will be important when we play someone other than the Browns. But hey we are learning how to win. It would be nice not to have to sit on the edge of my seat during the last few minutes of every game but I'll take 4-2 at this point.
I love what Campbell has done so far this year and I wish he wasn't hurt. But the good news is he isn't the driving force behind our offense. It's not like we need to replace an Aaron Rodgers. The key to the replacement is to minimize mistakes and let our weapons find ways to get the ball in the end zone. We are the one team in the league scoring a lot of points without gaudy stats from our QB. We have many ways to score as has been shown the last few weeks. And if we can bring in someone capable of completing a pass of more than 20 yards (which Campbell can't) then this could be a bonus. I'm nervous about Boller but the good thing about the situation is he has a couple of weeks of preseason to get ready as we play the Chiefs and Donkeys in our next 2 games. If he minimizes mistakes we'll be alright. But I hope the Raiders are looking for someone else.
Since: Apr 28, 2007
Posted on: October 16, 2011 10:37 pm
Jake Delhomme ?
Seriously, Jake is one of the worst qb's ever. He had one good playoff run and an exciting super bowl. That is it. Campbell had major flaws, but Jake flat out sucks. How good is Carolina with a rookie instead of that loser ? Unbelievable. Jake ruined several good teams and helped Fox get fired. I will puke if we sign him.
Carson Palmer for a conditional pick.
BTW, unless Hue Jackson learns how to manage a clock better, and stops passing on game winning FG's - it won't matter who the qb is.
On another note, stop running McFadden up the middle. This team will absolutely suck if he gets hurt !
Where is Derek Hagan ? Oh, that's right - Louis Murphy needs his snaps so he can continue to aligator arm every opp...............
One last thing, the Raiders cannot successfully convert 3rd and 1 in a power formation where Bush is the lead ball carrier. I do not know how many he has failed to convert, but it is way too many.
Concerned and Realistic Raider fan.
Since: Dec 6, 2006
Posted on: October 16, 2011 9:44 pm
Report: Campbell's dislocated shoulder ends year
What the heck is this guy talking about? How does a Raiders QB article lead to some guy talking about the Broncos and Teblow? Do you watch football? The Broncos suck with Orton and they suck a litlle less with Tebow. If you want to make comments you should find the section titled "little kid pony lovers" and post there, this area is reserved for grownups!


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Watto's Junk Yard / Re: Strange Magic« on: November 25, 2014, 10:03 PM »
Yeah, at first I hoped it was just overlay for the trailer, but nope, the characters are clearly singing. Repeatedly.
This section allows you to view all posts made by this member. Note that you can only see posts made in areas you currently have access to.
Wow, what's the point?
"Customer service." Treating customers like people instead of numbers. It's been pretty successful for Starbucks.


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Baldr Force EXE
- Episode Guide
S 1 : Ep 4
S 1 : Ep 3
S 1 : Ep 2
S 1 : Ep 1
First Contact
- show Description
Baldr Force EXE is an original video animation based on an action-shooter video game called Baldr Force. The OVA centers around an elite group of hackers and their leader Toru Soma, also known as Steppenwolf. Steppenwolf and his hacker cohorts earn their living by hacking information and selling it for profit. After hacking into a system, the hacker can operate a fighting machine known as a simulacrum. Steppenwolf and his hackers are about to disband after one last big job: hacking the military complex computers. They soon find this to be an electronic trap the military has sprung to trick cyber terrorists. The hackers then find themselves stuck in the middle of a battle between a vast army and ruthless terrorists. Many of Steppenwolf's friends are killed or captured, and he is forced to join the military he once stood so vehemently against.moreless
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