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A method for automated image analysis of a biological specimen by histological reconstruction. Also provided is an automated cell image method for analyzing a biological specimen that has consecutively been stained by either an in situ hybridization (ISH) method, or an immunohistochemistry (IHC) method or a nucleic acid stain, and counterstained. The method couples composite images in an automated manner for processing and analysis. To identify structure in tissue that cannot be captured in a single field of view image or a single staining technique, the disclosure provides a method for histological reconstruction to analyze many fields of view on potentially many slides simultaneously.
1. A method for automated image analysis of a sample, comprising:
automatically scanning one or more samples through an imaging field of an imaging system to obtain a plurality of different images that correspond to different position coordinates, each image having information of at least a portion of said one or more samples;
processing imaging signals from the imaging system to produce digital data of the different images;
digitally synthesizing the images to reconstruct a composite image which has data from the different images;
automatically detecting a target of interest on the sample by automatically measuring one or more parameters of the sample and automatically comparing the measured parameters against selected threshold values of the parameters for the target of interest; and
extracting information of the target of interest in the composite image.
2. The method as in
operating an imaging system at a first magnification to obtain a first set of the different images and a first reconstructed image;
setting the imaging system at a second magnification greater than the first magnification to obtain a second set of the different images and a second reconstructed image;
using the first composite image to identify a target therein; and
using the second composite image to extract desired information about the target.
3. The method as in
4. The method as in
5. A system for automated image analysis of a biological specimen, comprising:
(a) a slide handling mechanism that provides a biological sample to be analyzed;
(b) a microscope subsystem that
(1) automatically scans the sample at a plurality of different positions with different coordinates; and
(2) automatically obtains an image at each of said different coordinates to produce different images from different locations of the sample; and
(d) an image processor that
(1) automatically detects one or more targets of interest on the sample from the different images of the sample by automatically measuring one or more parameters of the sample and automatically comparing the measured parameters against selected threshold values of the parameters for the targets of interest;
(2) automatically reconstructs an image of a selected target on the sample from the obtained different images, wherein a reconstructed image of a selected target spreading in two or more spatially adjacent and different images of the sample is reconstructed by using image information from the spatially adjacent and different images; and
(3) processes the reconstructed image to obtain information of the selected target of interest.
6. The system of
7. The system of
automatically identify a coordinate of a candidate object or area of interest in the reconstructed image; and
automatically acquire a selected image of the object or area of interest, at the location coordinates obtained from the reconstructed image, wherein the selected image includes image information from two different images obtained at two different coordinates during the scanning.
8. The system of
receive from the microscope subsystem a first set of the different images and a first reconstructed image at a first magnification;
receive from the microscope subsystem a second set of the different images and a second reconstructed image at a second magnification greater than the first magnification;
use the first reconstructed image to identify a target therein; and
use the second reconstructed image to extract desired information about the target.
9. The system of
10. The system of
11. The system of
12. The system of
13. The system of
14. The system of
15. The system of
16. The system of
17. The system of
18. The system of
19. The system of
20. A system comprising:
(a) a slide handling mechanism that automatically loads and unloads a plurality of slide carriers to provide the system a biological sample for analysis;
(b) a microscope subsystem that automatically scans the sample through an imaging field to obtain a plurality of different images that correspond to different position coordinates, each image having information of at least a portion of the sample; and
(c) an image processor that automatically detects a target of interest on the sample from the different images of the sample by automatically measuring one or more parameters of the sample and automatically comparing the measured parameters against selected threshold values of the parameters for the target of interest.
21. The system of
an input hopper that has a guide key protruding from a side of the input hopper to align a keyway cutout of each of the slide carriers.
22. The system of
an unloading platform that holds the slide carriers after the scanning;
an output hopper that is configured to receive the slide carriers from the unloading platform; and
an unloading subassembly that rotates the unloading platform about a shaft, causing the slide carriers to be released into the output hopper.
23. The system of
24. A system for histological reconstruction of a sample, comprising:
(a) a slide feed mechanism that provides a sample of a biological specimen on a series of slides, wherein consecutive samples on said slides are:
(1) stained by a nuclear stain which includes hematoxylin and eosin (H/E), or
(2) detectably labeled by an immunohistochemistry (IHC) method and counterstained, or
(3) detectably labeled by an in situ hybridization (ISH) method and counterstained, or
(4) detectably labeled by a combined (IHC) and (ISH) method and counterstained;
(b) a microscope subsystem that automatically obtains a sample image from each of the samples by scanning the slides through an imaging field; and
(c) an image processor that
(1) automatically reconstructs the sample images to produce a single image frame in which a first sample image is paired with a consecutive sample having a corresponding image;
(2) automatically performs a histological reconstruction of the specimen; and
(3) automatically detects one or more targets of interest on the sample from the different sample images by automatically measuring one or more parameters of the sample associated with at least one of said stained and said labeled techniques in (a) and automatically comparing the measured parameters against selected threshold values of the parameters for the one or more targets of interest.
25. The system of
26. The system of
27. The system of
receive a first set of the sample images and a first single image frame from reconstructing the first set at a first magnification;
receive a second set of the sample images at a second magnification greater than the first magnification;
use the first single image frame to identify a target therein; and
use the second set of the samples images to extract desired information about the target.
This application is a continuation of U.S. patent applications Ser. No. 09/344,308, filed Jun. 24, 1999, and now U.S. Pat. No. 6,418,236 B1 which claims the benefit of priority under 35 U.S.C. �119(e) of U.S. provisional Application No. 60/129,384, filed Apr. 13, 1999. The aforementioned applications are explicitly incorporated herein by reference in their entirety and for all purposes.
The invention relates generally to light microscopy and, more particularly, to automated techniques of analyzing cytochemical and immunohistochemical staining.
In current cellular imaging systems, the area of a stained slide containing a stained cellular specimen is scanned by automated microscopy systems. The entire cellular specimen area of the slide is imaged with a series of field of view images. For further analysis, each field of view image must be separately analyzed to locate complete candidate objects of interest within the field of view image. This approach may be acceptable for cellular objects or clusters that are not so large that they extend beyond the field of view width or length of an image. Often, for both automated and manual analysis, only a single field of view is analyzed for morphological characteristics, so that the context of the analysis is limited to that field of view on a single slide.
A method for automated image analysis of a biological specimen is disclosed. A biological specimen is stained and counterstained for a specific marker or in the instance of immunohistochemistry or in situ hybridization, the marker is detectably labeled. Such labels include enzyme, radioisotopes, fluorescence or other labels well known in the art. The sample is then scanned at a plurality of positions by a photoimaging system to acquire an image. Adjacent positions are then used to reconstruct and provide a full picture image of the whole sample. A reconstructed whole sample image may then be further processed to identify coordinates that may have an object or area of interest. These coordinates are automatically selected for a candidate object of interest. In a preferred embodiment, a low magnification image of the candidate objects of interest is automatically obtained. Preferably the image is a color digital image. The candidate object of interest pixels in each sample are automatically morphologically processed to identify artifact pixels and the remaining candidate object of interest pixels in the sample not identified as artifact pixels. The apparatus obtaining the low magnification image is adjusted to a higher magnification, to acquire a higher magnification image of the sample, at the location coordinates corresponding to the low magnification image, for each candidate object or area of interest. Pixels of the higher magnification image in the first color space are automatically transformed to a second color space to differentiate higher magnification candidate objects of interest pixels from background pixels and identify, at higher magnification, objects of interest from the candidate object of interest pixels in the second color space. Thus, the pathologist or technician can identify whether the candidate object of interest has been specifically stained for the marker of interest, or counterstained, or both specifically stained and counterstained.
An automated cellular image method for analyzing a biological specimen, that has consecutively been stained with hematoxylin and eosin (H/E) on one tissue section and by one or several immunohistochemistry (IHC) and/or in situ hybridization (ISH) methods on parallel tissue sections, is also disclosed as a particular embodiment. To identify a structure in tissue that cannot be captured in a single field of view image or a single staining technique, the invention provides a method for histological reconstruction to analyze many fields of view on potentially many slides simultaneously. The method couples composite images in an automated manner for processing and analysis. A slide on which is mounted a biological specimen stained to identify structure of interest is supported on a motorized stage. An image of the biological specimen is generated, digitized, and stored. As the viewing field of the objective lens is smaller than the entire biological specimen, a histological reconstruction is made. These stored images of the entire tissue section may then be placed together in an order such that the H/E stained slide is paired with the immunohistochemistry slide so that analysis of the images may be performed simultaneously by the pathologist. The images may be superimposed or viewed as adjacent images.
In one embodiment, the invention provides a method for automated image analysis of a biological specimen by providing a biological sample to be analyzed, automatically scanning the sample at a plurality of coordinates, automatically obtaining an image at each of the coordinates, reconstructing an image of the sample from each individual image to create a reconstructed image and processing the reconstructed image to identify a candidate object or area of interest.
In another embodiment, the invention provides a method for histological reconstruction. In this method a sample of a biological specimen is divided into a number of subsamples. Each subsample is processed with a stain, counterstain, immunohistochemical technique, in situ hybridization technique, or a combination thereof. Each sample is then scanned and an image is obtained from each of the samples. The images are then reconstructed such that a first image is paired with a consecutive-corresponding sample image for identification of objects or areas of interest.
In yet another embodiment, the invention provides a computer program residing on a computer-readable medium, for automated image analysis of a biological specimen. The computer program comprises instructions for causing a computer to process a sample by scanning the sample at a plurality of coordinates, obtaining an image at each of the coordinates, reconstructing the sample from the individual images to create a reconstructed sample, identifying a coordinate of a candidate object or area of interest in the reconstructed image and acquiring an image at the coordinate obtained from the reconstructed image.
FIG. 1 is a perspective view of an apparatus for automated cell image analysis.
FIG. 2 is a block diagram of the apparatus shown in FIG. 1.
FIG. 3 is a plan view of the apparatus of FIG. 1 having the housing removed.
FIG. 4 is a side view of a microscope subsystem of the apparatus of FIG. 1.
FIG. 5 shows a slide carrier. FIG. 5a is a top view of a slide carrier for use levity the apparatus of FIG. 1. FIG. 5b is a bottom view of the slide carrier of FIG. 5a.
FIG. 6 shows views of an automated slide handling subsystem. FIG. 6a is a top view of an automated slide handling subsystem of the apparatus of FIG. 1. FIG. 6b is a partial cross-sectional view of the automated slide handling subsystem of FIG. 6a taken on line A—A.
FIGS. 7a-7 d illustrate the output operation of the automated slide handling subsystem.
FIG. 8 is a flow diagram of the procedure for automatically determining a scan area.
FIG. 9 is a block diagram of the microscope controller of FIG. 2
FIG. 10 shows a method of histological reconstruction.
An automated microscope for analyzing cellular specimens is shown in FIGS. 1 and 3 and in block diagram in FIG. 2. A motorized stage 38 may be used to support a slide 70 (FIG. 5). On the slide is mounted a biological specimen that is typically stained to identify a structure of interest for analysis. A biological specimen is defined as a cellular or acellular sample of biological origin, which has been mounted on a microscope slide. The biological specimen can be, for example, a tissue section, a biological fluid specimen, for example, a blood fraction cytospun on a microscope slide or a cell suspension directly seeded on a slide.
At least one optical sensing array, such as an objective lens, 44 a is located above the stage and a light source 48 is located beneath the stage. Light from the source illuminates the stage and slide so an image of the biological specimen is generated by the optical sensing array, for example an objective lens. This image is stored in memory. Preferably the image is a digitized or digital image. As the viewing field of the optical array is smaller than the entire biological specimen, the stage is moved in one planar direction by a distance that corresponds to the length of the field of view in that direction. The image generated at that position may then be captured and stored. The acquired image may be flipped along its centerline due to the optical flipping of the original image. Movement of the stage and capture of resulting image continue in the same direction until the end of the specimen area of the slide is reached. At that time, the stage is moved in the other planar direction by a distance that corresponds to the length of the field of view in that direction and another image is generated and stored. The slide is traversed or scanned in this manner until the entire specimen area of the slide has been viewed through the objective lens. These stored images may then be placed together in the order in which they were collected to generate a composite or reconstructed image of the cellular specimen. This composite image may then be analyzed to detect a structure that extends across more than one image field or more than one slide for further analysis. Such analysis may result in the identification of a candidate object or area of interest in both the field of view as well as objects that overlap two or more fields of view. In such instances, the system will automatically determine the coordinates for these candidate objects and may obtain additional images at various magnifications.
In existing cellular imaging systems, the area of the slide is scanned by automated microscopy systems to image the entire specimen area of the slide with a series of field of view images in the manner described above. However, these systems require that each field of view image is separately analyzed to locate complete candidate objects of interest within the field of view image for further analysis. This approach is acceptable for cellular objects that are not so large that they extend beyond the field of view width or length of an image. To identify structure in tissue that cannot be captured in a single field of view image, the present system uses an analysis method to identify field of view images that appear to contain part of a tissue structure to be analyzed. Field of view images so identified that are adjacent to one another are then identified as containing the tissue structure that the stain, antibody, or probe was intended to identify. This portion of the composite image may then be viewed under a higher magnification power for additional detail. The automatic building of a composite specimen image from the field of view images and the processing of the composite image or its constituent images to identify tissue structure that extends beyond one field of view is unknown.
In addition, a problem with existing automated systems is the continued need for operator input to initially locate cell objects for analysis. Such continued dependence on manual input can lead to errors including objects of interest being missed. These errors can be critical especially in assays for so-called rare events, e.g., finding one stained cell in a cell population of one million normal cells. Additionally, manual methods can be extremely time consuming and can require a high degree of training to properly identify or quantify cells. The associated manual labor leads to a high cost for these procedures in addition to the potential errors that can arise from long, tedious manual examinations. A need exists, therefore, for an improved automated cell image analysis system which can quickly and accurately scan large amounts of biological material on a slide.
Unless otherwise defined, 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
The term "nuclear stain" refers to a cytochemical stain that preferentially stains the nuclei of eukaryotic cells. Many nuclear stains are intercalating dyes. The term "intercalating dye" refers to a chemical compound that can insert itself in between adjacent nucleotides of a nucleic acid to provide a detectable color.
Many nuclear stains are known in the art, with one of the most commonly used being hematoxylin. Hematoxylin is often used in combination with various metallic salts (mordants). Hematoxylin stains are used for different staining purposes, and have a variety of colors, depending on the metal used. Aluminum lakes are purple to blue, depending on pH. Iron lakes are blue-black. Chromium lakes are blue-black. Copper lakes are blue-green to purple. Nickel lakes are various shades of violet. Tin lakes are red. Lead lakes are dark brown. Osmium lakes are greenish brown. Other nuclear stains include Giemsa stain, methyl green (which binds to AT-rich DNA regions), and Nuclear Fast-Red.
Fluorescent stains include Hoechst 33342; Hoechst 33258 (Calbiochem), a bisbenzimide DNA intercalator that excites in the near UV (350 nm) and emits in the blue region (450 nm); thiazole orange, a fluorogenic stain for DNA that excites in the blue region (515 nm) and emits in the green region (530 nm) of the visible spectrum; DAPI; ethidium bromide; propidium iodide; TOTO; YOYO-1; and SYTOX Blue or Green stains are also encompassed by the current invention. Several dyes either bind GC-rich or AT-rich chromosomal regions preferentially or show differences in fluorescence intensity upon binding those regions, yielding fluorescent banding patterns. For example, 7-Aminoactinomycin D binds selectively to GC-rich DNA regions and. 9-Amino-6-chloro-2-methoxyacridine fluoresces with greatest intensity in AT-rich DNA regions. Acridine homodimer fluoresces preferentially when bound to AT-rich DNA regions.
The term "counterstain," when used in combination with nuclear stains, refers to cytochemical stains that bind to a region of a eukaryotic cell other than the nucleus. Many counterstains are known in the art. One of the most common is eosin, which stains eukaryotic cell cytoplasm to varying shades of pink. Other counterstains are specific for a particular organelle or a protein in a cell. For example, the Kleihauer-Betke cytochemical stain is specific for hemoglobin F, a hemoglobin type preferentially expressed in fetal cells and therefore can be defined as a specific marker of fetal red blood cells.
The term "coordinate" or "address" is used to mean a particular location on a slide or sample. The coordinate or address can be identified by any number of means including, for example, X-Y coordinates, r-P coordinates, and others recognized by those skilled in the art.
In one embodiment, an automated cellular imaging method is used to identify fetal nucleated red blood cells in a maternal blood specimen. Fetal cells are first identified following the Kleihauer-Betke cytochemical stain for hemoglobin F. Fetal cells are identified by the automated cellular imaging system as objects on the basis of their bright red color (indicative of Hemoglobin F) as compared to maternal red blood cells. To assure that appropriate objects are identified, size and shape morphological "filters" are used to exclude very small and very large objects.
Cells are counterstained with an additional cytochemical stain for nucleic acids, resulting in a blue color for nucleated red blood cells (generally, fetal red blood cells). An automated image analysis system identifies blue objects of the appropriate size and shape for an erythrocyte nucleus among the bright red objects, allowing the imaging system to identify and enumerate nucleated fetal red cells. Such cells can be enumerated, allowing for a screen for Down's syndrome in the fetus, wherein the frequency of such cells is typically higher in Down's syndrome pregnancies compared with normal pregnancies.
In a preferred embodiment, the slides are stained with hematoxylin/eosin (H/E) and one or several parallel slides containing adjacent sections are stained for one or several specific markers.
The results of the H/E staining provide cells with nuclei stained blue-black, cytoplasm stained varying shades of pink; muscle fibers stained deep pinky red; fibrin stained deep pink; and red blood cells stained orange-red.
For example, hematoxylin/eosin (H/E) slides are prepared with a standard H/E protocol. Standard solutions include the following: (1) Gills hematoxylin (hematoxylin 6.0 g; aluminum sulphate 4.2 g; citric acid 1.4 g; sodium iodate 0.6 g; ethylene glycol 269 ml; distilled water 680 ml); (2) eosin (eosin yellowish 1.0 g; distilled water 100 ml); (3) lithium carbonate 1% (lithium carbonate 1 g; distilled water 100 g); (4) acid alcohol 1% 70% (alcohol 99 ml conc.; hydrochloric acid 1 ml); and (5) Scott's tap water. In a beaker containing 1 L distilled water, add 20 g sodium bicarbonate and 3.5 g magnesium sulphate. Add a magnetic stirrer and mix thoroughly to dissolve the salts. Using a filter funnel, pour the solution into a labeled bottle.
The staining procedure is as follows: (1) Bring the tissue or cell sections to water; (2) place sections in hematoxylin for 5 minutes (min); (3) wash in tap water; (4) 'blue' the sections in lithium carbonate or Scott's tap water; (5) wash in tap water; (6) place sections in 1% acid alcohol for a few seconds; (7) wash in tap water; (8) place sections in eosin for 5 min; (9) wash in tap water; and (10) dehydrate with graded alcohol solution.
A specific marker is a molecule or a group of molecules, which is present in only a subset of the components of a biological specimen and therefore identifying specifically the components having the marker. Specific markers are frequently defined as antigens recognized by specific antibodies (monoclonals or polyclonals) and can be detected by immunohistochemistry. Another group of specific markers is defined by the capacity of these markers to hybridize, specifically, a nucleic acid probe. These markers can usually be detected by in situ hybridization. A third group of specific markers can be defined by their enzymatic activity and can be detected by histochemistry. A fourth group of specific markers can be stained directly, histochemically, using a specific dye.
A fifth group of specific markers can be defined as being receptors binding specifically to one or several ligands. A specific ligand is itself used for the detection of the receptor-ligand complex, using a detection method involving either histochemistry, or immunohistochemistry or in situ hybridization.
Immunohistochemical techniques as used herein encompasses the use of reagents detecting cell specific markers, such reagents include, for example, as antibodies and nucleic acids probes. Antibodies, including monoclonal antibodies, polyclonal antibodies and fragments thereof, are often used to identify proteins or polypeptides of interest in a sample. A number of techniques are utilized to label objects of interest according to immunohistochemical techniques. Such techniques are discussed in Current Protocols in Molecular Biology, Unit 14 et seq., eds. Ausubel, et al., John Wiley & Sons, 1995, the disclosure of which is incorporated herein by reference. For example, the following procedure is an example of immunohistochemical staining using an antibody recognizing, specifically, the HER2 protein. HER2 overexpression has been described as a specific marker in a high percentage of breast cancer carcinomas. This protocol is intended for staining a paraffin embedded tissue section prepared according to a conventional procedure.
The section is deparaffinized using two baths of xylene and rehydrated through graded alcohols baths and finally in deionized water. The section is then incubated with an Antigen Retrieval Buffer, containing Citrate, for 40 minutes at 95� C. The slide is then cooled down at room temperature for 20 minutes in the same buffer. The slide is then rinsed in deionized water.
The area surrounding the tissue section is carefully dried and a hydrophobic delimiting pen is used to draw a line around the specimen, on the glass slide. A peroxidase blocking solution is added on the section and incubated 5 minutes at room temperature. After being washed twice with the Wash Buffer (a balanced salt solution), the tissue section is incubated 30 minutes at room temperature, with the primary antibody recognizing the HER2 protein.
After 3 washes with the Wash Buffer, the tissue section is incubated with the peroxidase conjugated secondary antibody. The secondary antibody will recognize specifically the primary antibody. The slide is then washed 3 times with the Wash Buffer. Then the tissue section is incubated in presence of DAB and hydrogen peroxide for 10 minutes, before being washed with water.
The tissue section is counterstained in Hematoxylin for 2 minutes and rinsed again with water. The slide is mounted with a cover-slip using an aqueous mounting medium.
Immunohistochemical localization of cellular molecules uses the ability of antibodies to bind specific antigens, for example proteins of interest such as onco-proteins and enzymes, with high affinity. These antibodies can be used to localize antigens to subcellular compartments or individual cells within a tissue.
In situ hybridization techniques include the use of specifically labeled nucleic acid probes, which bind to cellular RNA or DNA in individual cells or tissue section. Suitable nucleic acid probes may be prepared using standard molecular biology techniques including subcloning, plasmid preparation, and radiolabeling or non-radioactive labeling of the nucleic acid probe.
In situ hybridization is often performed on either paraffin or frozen sections. Such techniques often include fine sectioning of tissues to provide samples that are only a single to a few cell layers thick. For example paraffin blocks containing a tissue sample are cut into thin, approximately 8 um tissue sections, which are subsequently mounted on subbed slides to be further processed for in situ hybridization. Alternatively, methacrylate may be used for sectioning. Cryosectioning techniques are particularly suitable for immunohistochemistry and enzyme histochemistry.
Immunofluorescent labeling of a tissue section often use a sandwich assay or a primary antibody and secondary antibody-fluorochrome conjugate. Slides containing a tissue section of interest are washed in phosphate buffered saline and then exposed to a primary antibody which will bind to the protein object of interest. Subsequently the slides are washed and exposed to the secondary antibody which binds to the first or primary antibody. The slide is washed and then developed. Numerous other techniques well known in the art of immunohistochemical staining and in situ hybridization are easily adaptable for use in immunohistochemical reconstruction as disclosed herein.
Thus, a combination of techniques using both chemical staining and/or immunohistochemical and/or in situ hybridization may be used in the present methods. For example, numerous subsamples may be prepared from a single tissue specimen. A first subsample may be chemically stained as discussed above, and a subsequent subsample may be subjected to immunohistochemical and in situ hybridization techniques. Each subsamples is scanned and processed as discussed below. Reconstructed images may then be overlapped or processed to further identify objects of interest.
Histological reconstruction is a process whereby an image of a whole specimen is constructed from analyzed pieces of the specimen, particularly when the biological specimen has been mounted on a slide. This image is created by piecing together more than one field of view at any particular magnification.
With reference to FIG. 10, an image 302, representing an objective's field of view is acquired at a first particular coordinate on the slide sample 301. The slide is automatically repositioned on the X-Y stage to obtain a new or second field of view corresponding to a second particular coordinate 302. This new field of view is preferably immediately adjacent to the first field of view, however, so long as the coordinates, thus the address/identity, of each field of view are retained in the imaging system, histological reconstruction may be performed. This process is repeated until images for the whole of the specimen have been acquired.
Based upon each image's X and Y coordinate, the specimen is digitally reconstructed. As part of the reconstruction, the image may be flipped to correct for the optical flipping of the original image.
The process of forming a histological reconstructed image involves having the apparatus scan a microscope slide of interest, and form the image that constitutes a reconstruction of the images taken during the scan. The image that is formed can be a full-color reconstruction of the entire scan area, or a fraction of the whole scan area, for example, reconstruction of the entire scan area that identifies objects or areas of interests. The reconstructed digital image can then be used for further processing or analysis to identify previously undetected objects or areas of interest. For example, objects or areas of interest overlapping one or more fields of view or slides may thus be identified in the reconstructed digital image.
With reference to FIGS. 1 and 2, the apparatus 10 also referred to as the system, comprises a microscope 32 with a motorized X, Y and Z stage 38, a camera 42, a computer 22 adapted to receive and process video images, and a set of software programs to control the apparatus and to execute the method. A measurement of the optical properties of the sample features is used to form an image of the scannable area of the slide, to find sub-regions of interest, and to analyze the properties of these regions. The image processing method that evaluates the sample to find regions of interest uses a measure of the hue, saturation or intensity and luminosity of a 24-bit color image to produce a white on black target image of interest. This image is processed by separately converting the full color image to components of hue, saturation or intensity and luminosity, thresholding the components, and performing a logical "AND" between the two images, then thresholding the resulting image such that any pixel value above zero becomes 255. The processing and image acquisition will be further understood with reference to the apparatus described below.
The invention provides a method for automated analysis of a biological specimen, which eliminates the need for operator input to locate biological objects or areas of interest for analysis.
With reference to FIG. 1, a slide prepared with a biological specimen and reagent is placed in a slide carrier 60 (FIG. 5) which preferably holds four slides. The slide carriers are loaded into an input hopper 16 of the automated system 10. The operator then enters data identifying the instrument protocol which contains information on the size, shape and location of a scan area on each slide, or, preferably, the system automatically locates a scan area for each slide during slide processing. The operator then activates the system 10 for slide processing. At system activation, a slide carrier 60 is positioned on an X-Y stage 33 of an optical system, such as microscope subsystem 32. Any bar codes used to identify slides are read and stored for at a low magnification, typically 10�. At each location of the scan, a low magnification image is acquired and processed to detect candidate objects or areas of interest, preferably, color, size and shape are used to identify objects or areas of interest. The location of each candidate object or area of interest may be stored by reference to its coordinates or address. Each field of view may also be stored as part of a larger composite image (described in detail elsewhere).
At the completion of the low level scan for each slide in the carrier on the stage, the optical system may be adjusted to a higher magnification such as 40� or 60�, for additional specimen processing and image acquisition, and the X-Y stage is positioned to the stored locations for the candidate objects or areas of interest on each slide in the carrier. A higher magnification image is acquired for each candidate object or area of interest and a series of image processing steps are performed to confirm the analysis, which was performed at low magnification. A higher magnification image is stored for each continued object or area of interest. These images are then available for retrieval by a pathologist or cytotechnologist to review for final diagnostic evaluation. Having stored the location of each object or area of interest, a mosaic comprising the candidate objects or areas of interest for a slide may be generated and stored. The pathologist or cytotechnologist may view the mosaic or may also directly view the slide at the location of an object or area of interest in the mosaic for further evaluation. The mosaic may be stored on magnetic or optical media for future reference or may be transmitted to a remote site for review or storage. The entire process involved in examining a single slide takes on the order of 4-100 min depending on scan area size and the number of detected candidate objects of interest.
The invention has utility in prenatal diagnosis of fetal cells, rare event detection, rapid cell counting, tissue evaluation, and other diagnoses.
The processing of images acquired in the automated scanning preferably includes the steps of transforming the image to a different color space, here preferably by hue, saturation and intensity. The pixels of the filtered image are dynamically thresholded to suppress background material; performing a morphological function to remove artifacts from the thresholded image; analyzing the thresholded image to determine the presence of one or more regions of connected pixels having the same color; and categorizing every region having a size greater than a minimum size as a candidate object or area of interest.
According to another aspect, the scan area is automatically determined by scanning the slide; acquiring an image at each slide position; analyzing texture or color information for each image to detect the edges of the specimen and storing the locations corresponding to the detected edges to define the scan area.
According to yet another aspect, automated focusing of the optical system is achieved by initially determining a focal surface from an array of points or locations in the scan area. The derived focal surface enables subsequent rapid automatic focusing in the low power scanning operation. In one embodiment, the focal plane is determined by determining proper focal positions across an array of locations and performing a least squares fit of the array of focal positions to yield a focal plane across the array. Preferably, a focal position at each location is determined by incrementing the position of a Z stage for a fixed number of coarse and fine iterations. At each iteration, an image is acquired and a pixel variance, morphological gradient or other optical parameter about a pixel mean for the acquired image is calculated to form a set of evaluation data. The peak value of the least squares fit curve is selected as an estimate of the best focal position.
In another aspect, a focal position method for a higher magnification locates a region of interest centered about a candidate object of interest within a slide which was located during an analysis of the low magnification images. The region of interest is preferably n columns wide, where n is a power of 2. The pixels of this region are then processed using a Fast Fourier Transform to generate a spectra of component frequencies and corresponding complex magnitude for each frequency component. Preferably, the complex magnitude of the frequency components which range from 25% to 75% of the maximum frequency component are squared and summed to obtain the total power for the region of interest. This process is repeated for other Z positions and the Z position corresponding to the maximum total power for the region of interest is selected as the best focal position. This focal method can be used with many stains and types of cellular specimens.
According to still another aspect, a method for automated slide handling is provided. A slide is mounted onto a slide carrier 60 (FIG. 5) with a number of other slides side-by-side. The slide carrier 60 is positioned in an input feeder 16 with other slide carriers to facilitate automatic analysis of a batch of slides. The slide carrier is loaded onto the X-Y stage 38 of the optical system 32 for the analysis of the slides thereon. Subsequently, the first slide carrier is unloaded into an output feeder 18 after automatic image analysis and the next carrier is automatically loaded.
Referring to the FIGURES, an apparatus for automated cell image analysis of biological specimens is generally indicated by reference numeral 10 as shown in perspective view in FIG. 1 and in block diagram form in FIG. 2. The apparatus 10 comprises a microscope subsystem 32 housed in a housing 12. The housing 12 includes a slide carrier input hopper 16 and a slide carrier output hopper 18. A door 14 in the housing 12 secures the microscope subsystem from the external environment. A computer subsystem comprises a computer 22 having two system processors 23, an image processor 25 and a communications modem 29. The computer subsystem further includes a computer monitor 26 and an image monitor 27 and other external peripherals including storage device 21, pointing device 30, keyboard 28 and color printer 35. An external power supply 24 is also shown for powering the system. Viewing oculars 20 of the microscope subsystem project from the housing 12 for operator viewing. The apparatus 10 further includes a 3-chip CCD camera 42 for acquiring images through the microscope subsystem 32. The computer directly controls a number of microscope subsystem functions described further in detail.
An automatic slide feed mechanism 37 in conjunction with X-Y stage 38 provide automatic slide handling in the apparatus 10. An illumination light source 48 projects light onto the X-Y stage 38 which is subsequently imaged through the microscope subsystem 32 and acquired through the 3-chip CCD camera 42 for processing by the image processor 25. A Z stage or focus stage 46 under control of the microscope controller 31 provides displacement of the microscope subsystem in the Z plane for focusing. The microscope subsystem 32 further includes a motorized objective turret 44 for selection of objectives.
The apparatus 10 is for the unattended automatic scanning of prepared microscope slides for the detection and counting of candidate objects or areas of interest, such as stained cells. In one embodiment the invention may be utilized for tissue analysis. In another embodiment, rare event detection in which there may be only one candidate object of interest per several hundred thousand normal cells, e.g., one to five candidate objects of interest per 2 square centimeter area of the slide. The apparatus 10 automatically locates and counts candidate objects or areas of interest and estimates normal cells present in a cellular specimen on the basis, for example, of color, size and shape characteristics. A biological specimen may be prepared with a reagent to obtain a colored insoluble precipitate. The apparatus, in one embodiment, is used to detect this precipitate as a candidate object or area of interest.
During operation of the apparatus 10, a pathologist or laboratory technician mounts prepared slides onto slide carriers. A slide carrier 60 is illustrated in FIG. 5 and is described further below. Each slide carrier holds up to 4 slides. Up to 25 slide carriers are then loaded into input hopper 16. The operator can specify the size, shape and location of the area to be scanned or alternatively, the system can automatically locate this area. The operator then commands the system to begin automated scanning of the slides through a graphical user interface. Unattended scanning begins with the automatic loading of the first carrier and slide onto the motorized X-Y stage 38. A bar code label affixed to the slide is read by a bar code reader 33 during this loading operation. Each slide is then scanned at a user selected low microscope magnification, for example, 10�, to build a histological reconstruction or identify candidate objects based on their color, size and shape characteristics. The X-Y locations of candidate objects or areas of interest are stored until scanning is completed.
After the low magnification scanning is completed, the apparatus may automatically return to each candidate object or area of interest, if necessary, reimaging and refocusing at a higher magnification such as 40� and performs further analysis to confirm the biological candidate. The apparatus stores an image of the object or area of interest for later review by a pathologist. All results and images can be stored to a storage device 21 such as a removable hard drive or optical disc or DAT tape or transmitted to a remote site for review or storage. The stored images for each slide can be viewed in a mosaic of images for further review. In addition, the pathologist or operator can also directly view a detected object or area of interest through the microscope using the included oculars 20 or on image monitor 27.
The two system processors 102 further control an illumination controller 106 for control of substage illumination 48. The light output from, for example, a halogen light bulb, which supplies illumination for the system, can vary over time due to bulb aging, changes in optical alignment, and other factors. In addition, slides which have been "over stained" can reduce the camera exposure to an unacceptable level. To compensate for these effects, the illumination controller 106 is included. This controller is used in conjunction with light control software to compensate for the variations in light level. The light control software samples the output from the camera at intervals (such as between loading of slide carriers), and commands the controller to adjust the light level to the desired levels. In this way, light control is automatic and transparent to the user and adds no additional time to system operation.
The system processors 23 are preferably comprised of dual parallel Intel Pentium Pro 400 MHZ devices. The image processor 25 is preferably a Matrox Genesis board. The computer, in a preferred embodiment, operates under Windows NT. It will be recognized that any number of processors and operating systems can be used in the methods and in conjunction with the present invention.
Referring now to FIGS. 3 and 4, further detail of the apparatus 10 is shown. FIG. 3 shows a plan view of the apparatus 10 with the housing 12 removed. A portion of the automatic slide feed mechanism 37 is shown to the left of the microscope subsystem 32 and includes slide carrier unloading assembly 34 and unloading platform 36 which in conjunction with slide carrier unloading hopper 18 function to receive slide carriers which have been analyzed.
Vibration isolation mounts 40, shown in further detail in FIG. 4, are provided to isolate the microscope subsystem 32 from mechanical shock and vibration that can occur in a typical laboratory environment. In addition to external sources of vibration, the high speed operation of the X-Y stage 38 can induce vibration into the microscope subsystem 32. Such sources of vibration can be isolated from the electro-optical subsystems to avoid any undesirable effects on image quality. The isolation mounts 40 comprise a spring 40 a and piston 40 b submerged in a high viscosity silicon gel which is enclosed in an elastomer membrane bonded to a casing to achieve damping factors on the order of 17% to 20%.
The automated slide handling subsystem operates on a single slide carrier at a time. A slide carrier 60 is shown in FIGS. 5a & 5 b, which provide a top view and a bottom view respectively. The slide carrier 60 includes up to four slides 70 mounted with adhesive tape 62. The carrier 60 includes ears 64 for hanging the carrier in the output hopper 18. An undercut 66 and pitch rack 68 are formed at the top edge of the slide carrier 60 for mechanical handling of the slide carrier. A keyway cutout 65 is formed in one side of the carrier 60 to facilitate carrier alignment. A prepared slide 72 mounted on the slide carrier 60 includes a sample area 72 a and a bar code label area 72 b.
FIG. 6a provides a top view of the slide handling subsystem which comprises a slide input module 15, a slide output module 17 and X-Y stage drive belt 50. FIG. 6b provides a partial cross-sectional view taken along line A—A of FIG. 6a.
The slide input module 15 includes a slide carrier input hopper 16, loading platform 52 and slide carrier loading subassembly 54. The input hopper 16 receives a series of slide carriers 60 (FIGS. 5a and 5 b) in a stack on loading platform 52. A guide key 57 protrudes from a side of the input hopper 16 to which the keyway cutout 65 (FIG. 5a) of the carrier is fit to achieve proper alignment.
The input module 15 further includes a revolving indexing cam 56 and a switch 90 mounted in the loading platform 52, the operation of which is described further below. The carrier subassembly 54 comprises an infeed drive belt 59 driven by a motor 86. The infeed drive belt 59 includes a pusher tab 58 for pushing the slide carrier horizontally toward the X-Y stage 38 when the belt is driven. A homing switch 95 senses the pusher tab 58 during a revolution of the belt 59.
Referring specifically to FIG. 6a, the X-Y stage 38 is shown with x position and y position motors 96 and 97 respectively which are controlled by the microscope controller 31 (FIG. 9) and are not considered part of the slide handling subsystem. The X-Y stage 38 further includes an aperture 55 for allowing illumination to reach the slide carrier. A switch 91 is mounted adjacent the aperture 55 for sensing contact with the carrier and thereupon activating a motor 87 to drive stage drive belt 50 (FIG. 6b). The drive belt 50 is a double-sided timing belt having teeth for engaging pitch rack 68 of the carrier 60 (FIG. 5b).
The slide output module 17 includes slide carrier output hopper 18, unloading platform 6, and slide carrier unloading subassembly 34. The unloading subassembly 34 is a motor 89 for rotating the unloading platform 36 about shaft 98 during an unloading operation described further below. An outfeed gear 93 driven by motor 88 rotatably engages the pitch rack 68 of the carrier 60 (FIG. 5b) to transport the carrier to a rest position against switch 92. A spring loaded hold-down mechanism holds the carrier in place on the unloading platform 36.
The slide handling operation is now described. Referring to FIG. 7, a series of slide carriers 60 are shown stacked in input hopper 16 with the top edges 60 a aligned. As the slide handling operation begins, the indexing cam 56 driven by motor 85 advances one revolution to allow only one slide carrier to drop to the bottom of the hopper 16 and onto the loading platform 52.
FIGS. 7a-7 d show the cam action in more detail. The indexing cam 56 includes a hub 56 a to which are mounted upper and lower leaves 56 b and 56 c respectively. The leaves 56 b & 56 c are semicircular projections oppositely positioned and spaced apart vertically. In a first position shown in FIG. 8a, the upper leaf 56 b supports the bottom carrier at the undercut portion 66. At a position of the indexing cam 56 rotated 180�, shown in FIG. 7b, the upper leaf 56 b no longer supports the carrier and instead the carrier has dropped slightly and is supported by the lower leaf 56 c. FIG. 8c shows the position of the cam 56 rotated 270� wherein the upper leaf 56 b has rotated sufficiently to begin to engage the undercut 66 of the next slide carrier while the opposite facing lower leaf 56 c still supports the bottom carrier. After a full rotation of 360� as shown in FIG. 7d, the lower leaf 56 c has rotated opposite the carrier stack and no longer supports the bottom carrier which now rests on the loading platform 52. At the same position, the upper leaf 56 b supports the next carrier for repeating the cycle.
Referring again to FIGS. 6a & 6 b, when the carrier drops to the loading platform 52, the contact closes switch 90 which activates motors 86 and 87. Motor 86 drives the infeed drive belt 59 until the pusher tab 58 makes contact with the carrier and pushes the carrier onto the X-Y stage drive belt 50. The stage drive belt 50 advances the carrier until contact is made with switch 91, the closing of which begins the slide scanning process described further herein. Upon completion of the scanning process, the X-Y stage 38 moves to an unload position and motors 8, and 88 are activated to transport the carrier to the unloading platform 36 using stage drive belt 50. Motor 88 drives outfeed gear 93 to engage the carrier pitch rack 68 of the carrier 60 (FIG. 5b) until switch 92 is contacted. Closing switch 92 activates motor 89 to rotate the unloading platform 36.
The unloading operation is shown in more detail in end views of the output module 17 (FIGS. 7a-7 d). In FIG. 7a, the unloading platform 36 is shown in a horizontal position supporting a slide carrier 60. The hold-down mechanism 94 secures the carrier 60 at one end. FIG. 7b shows the output module 17 after motor 89 has rotated the unloading platform 36 to a vertical position, at which point the spring loaded hold-down mechanism 94 releases the slide carrier 60 into the output hopper 18. The carrier 60 is supported in the output hopper 18 by means of ears 64 (FIGS. 5a and 5 b). FIG. 7c shows the unloading platform 16 being rotated back towards the horizontal position. The platform 36 rotates upward and contacts the deposited carrier 60. The upward movement pushes the carrier toward the front of the output hopper 18. FIG. 7d shows the unloading platform 36 at its original horizontal position after having output a series of slide carriers 60 to the output hopper 18.
The aspects of the apparatus 10 relating to scanning, focusing and image processing are further described in patent application Ser. No. 08/758,436 which is incorporated herein.
Aspects of the invention may be implemented in hardware or software, or a combination of both. However, preferably, the algorithms and processes of the invention are implemented in one or more computer programs executing on programmable computers each comprising at least one processor, at least one data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described herein and generate output information. The output information is applied to one or more output devices, in known fashion.
Each program may be implemented in any desired computer language (including machine, assembly, high level procedural, or object oriented programming languages) to communicate with a computer system. In any case, the language may be a compiled or interpreted language.
Each such computer program is preferably stored on a storage media or device (e.g., ROM, CD-ROM, tape, or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The inventive system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
A number of embodiments of the present invention have been described. Nevertheless, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not to be limited by the specific illustrated embodiment, but only by the scope of the appended claims.
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text:
Hold down Control (PC) or Command (Mac) key + mouseclick to select more than one option
|Avg. Dinner Entrée
|Greater than $25
|$18.01 - $25
|$12.01 - $18
|$7.01 - $12
|Less than $7
Posted by anonymous on 03/21/2013
In a neighborhood of few decent choices, Main Noodle House offers a solid fresh meal and very good service. Delivery orders are prompt and accurate. Excellent sautéed Chinese broccoli and won-ton soup.
Posted by Tom on 01/13/2013
I can't believe how many poor reviews this restaurant received! I have attempted to find a good chinese spot in manhattan and this is the place to go. I have recommended this restaurant to many colleagues of mine and I will certainly continue to eat here! The location is very convenient to get to and the food is definitely good quality.
Posted by anonymous on 11/15/2012
The food here is not the best Chinese food I've had, but is good. I ordered the chicken lo mein lunch dish, which was delicious. It came with wonton soup, which was not so good (only 1 wonton in a whole pint of broth), and the brown rice was cold. What bothered me the most was the service I got while ordering on the phone. The woman was rude, rushed my order, and hung up on me while I was in mid sentence without saying thank you. Then when I went to pickup my order, the same lady was on the phone with another customer, and I couldn't believe how rude she was to this customer. I will most likely not order from this place again.
Posted by anonymous on 11/05/2012
I'm from Brazil and I've stayed at Marriott's Residence Inn which is quite close to the restaurant. At my first evening in NY, after travelling 10 hours, my family and I were very tired so we went for a fast meal in some walking distance place. We ate at Main Noodles House and it was fantastic! I ate the "Lo Mein" and my parents a spicy chicken. Almost everyday we came back for some noodles. Now, everytime we go to NY we say we are going for some "Lo Mein"!
Posted by rukhsaar on 07/05/2012
foods great but service is ehh, i dont like repeating my info and staying on hold though. but overall its okay =)
Posted by S.C. on 06/04/2012
My friend took me here for a lunch as we both work in the area. I was very impressed with the food and service, so much so that I returned the following week with my bf for lunch. The food is presented nicely in serving dishes with plates for sharing. The restaurant is small and tho always packed (a good sign!) we were very comfortable. The service was quick and attentive. The food (general tso's tofu, chicken lo mein, eggplant ans chicken) was very tasty a great value with all under $8 for lunch including soup or eggroll. I never sit down for lunch, but this will definitely become a regular routine for me now. I'm craving it right now!
Posted by Anonymous on 02/13/2012
Thanks to menu pages found this place while shopping in the fashion district... We will be back! Lunch specials are great, and the service is so fast!

subset: commoncrawl


text:
Day of Caring and Giving
Updated: 08/06/2004 - The United Way of East Mississippi got hands on help from community volunteers in its annual "Day of Caring".
Updated: 08/06/2004 - The United Way of East Mississippi got hands on help from community volunteers in its annual "Day of Caring".
Updated: 08/06/2004 - Newton Public School students hit the books again Friday as they began the 2004-2005 year.
Updated: 08/06/2004 - Authorities say a disgruntled former Mississippi prison inmate is accused of plotting to blow up the federal courthouse in downtown Chicago.
Updated: 08/06/2004 - If you are looking to build or buy a home but you don't think you can afford it, you might be in luck!
Updated: 08/06/2004 - There's good news for customers of the Long Creek Water Association.
Updated: 08/05/2004 - A Civil Rights bus tour is continuing its journey that will end up in Philadelphia, Miss., on Aug. 20.
Updated: 08/05/2004 - A woman involved in a wreck in Meridian Wednesday died hours later at a local hospital.
Updated: 08/05/2004 - As school starts in Enterprise, school officials say their biggest challenge and number one goal is continuing to grow and be as far above average as possible.
Updated: 08/05/2004 - With only about 1,200 students and three schools, the city of Philadelphia's school district is small. However, it's reaching for higher goals.
Updated: 08/05/2004 - Saturn is recalling more than 246,000 VUE sport utility vehicles, because of concerns about the rear suspension.
Updated: 08/05/2004 - Thursday marked a major milestone for one of Meridian's most noted native sons.
Updated: 08/04/2004 - As students head back to class, the Meridian Police Department will be out in full force. They will be making sure your kids are safe from speeders in school zones.
Updated: 08/04/2004 - The Meridian Police Department is investigating a home invasion robbery that happened Tuesday night.
Updated: 08/04/2004 - East Central Community College in Decatur has announced its annual Alumni Association Awards.
Updated: 08/04/2004 - The Parents as Teachers Program of Meridian Public Schools hosted a health fair for parents, students and the community.
Updated: 08/04/2004 - Students attending Poplar Springs Elementary School, along with their parents, took an advance look at the campus during open house.
Updated: 08/04/2004 - An arrest has been made in the weekend stabbing death of a Kemper County man.
Updated: 08/04/2004 - A Meridian man has been sentenced to 15 years in prison after pleading guilty to an aggravated DUI charge in Oktibbeha County.
Updated: 08/03/2004 - The Justice Department says President Bush's appointment of former Alabama attorney general Bill Pryor as a federal judge is constitutional.
Updated: 08/03/2004 - The Clarke County Sheriff's Department is pulling out of the East Mississippi Drug Task Force, but not quitting its fight against illegal drugs.
Updated: 08/03/2004 - A major tree planting effort may be taking place soon on Meridian's 22nd Avenue. The goal is enhance the beauty of that entry point into the city.
Updated: 08/03/2004 - When it comes to how well your child does in school, a good teacher can make all the difference.
Updated: 08/03/2004 - West Alabama authorities are asking for your help in locating a suspect in a Livingston store robbery.
Updated: 08/03/2004 - Mississippi Cong. Gene Taylor held a Town Hall meeting in Enterprise Monday night.
Updated: 08/02/2004 - Clarke County will halt its participation in the East Mississippi Drug Task Force at the end of the fiscal year.


text:
Arizona Gov. Jan Brewer reconsiders and signs embalmer intern bill
PHOENIX — Arizona's 51 crematories and 163 funeral homes will be able to
hire intern trainees with just 16 hours of training now that Gov. Jan Brewer has
signed off on the proposal.
Brewer vetoed House Bill 2271 last month and said the 8 hours of training it
required wasn't enough. She worried about the consequences if funeral homes
employed intern trainees with inadequate training and minimal classroom work.
Republican Rep. Tom Forese of Chandler revived the bill by adding it to Senate
Bill 1342 and doubling the training. Brewer signed it Thursday.
The trainee can help prepare human remains, help with embalming bodies and
perform any task a licensed intern is currently allowed to do.
Forese says people need to work in the industry before attending a mortuary
science college.


text:
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text: job interviews - job interviews questions and answers, job ...WebAssignwww.aplia.comOver 5,300 instructors at more than 1,700 institutions have used Aplia.CURRENT AFFAIRS 2012 TRIVIA QUIZ CURRENT AFFAIRS 2011 PDF C# Interview Questions and Answers CSHARP Dot Net Interview Quiz ...Web Dev Support - Web Dev Help from Web Dev Expertsapplications and output. - School Improvement in Marylandugcenglish.commultiple-choice-questions-for-ugc-net-english-quizmultiple choice questions for ugc net english. Latest syllabus questions accoring to UGC English. UGC NET English multiple choice questions for the 2nd and 3rd paper.Multiple Choice Questions for UGC NET English Quiz UGC www.artographed.comlogos-quiz-game-answers-android-level-1Logos Quiz Answers - Level 1 This is Logos Quiz Answers Game Level 1 in Carlos Alcarrias Version. You might find some part of this level quite difficult therefore ...www.armstrong.eduScienceandTechnologyiceicewelcomeWelcome. With computers and technology affecting most aspects of society, students in Computer Science and Information Technology, learn skills and talents that are ...teach-nology.com - Worksheets, Lesson Plans, Teacher Resources ...www.treeknox.comtechnical-questionsdotnetcsharp.netThese questions are frequently asked in all IT Companies like 3i infotech,Cts,Tcs,Wipro,Accenture,Amazon,Aspire,BlueStar,Flextronics,Honeywell,Calsoft,Capgemini ...Register at FaaDoOEngineers.commdk12.org...vsctechnologyliteracystandards.pdfnbsp#0183#32PDF fileMaryland Technology Literacy Standards for Students Standard 1.0 Technology Systems Develop foundations in the understanding and uses of technology systemscurrent-affairs-quiz-questionsanswers.blogspot.com...2011-pdf-free...Feb 20, 2011nbsp#0183#32This is the Current affairs quiz blog, fifa quiz, INDIA FREEDOM QUIZ INDIA INDEPENDENCE QUIZ, and Bank preparation, current affairs of kerala and www.faadooengineers.comregister.phpTechnical FestivalsAll Technical Festivals across IITs, NITs, IIITs amp other private engineering colleges in India are covered in this section.www.experts-exchange.comnbsp#8250#32Web DevelopmentWeb Dev help from the top Web Dev experts in the world is minutes away. Whether asking a question or searching previous answers, youre sure to find all the Web Dev ...Aplia Engage. Prepare. Educate.www.teach-nology.comWorksheetsnbsp#0183#32Reading Worksheetsnbsp#0183#32Printablesnbsp#0183#32Math Worksheetsnbsp#0183#32RubricsTeach-nology.com offers teachers FREE access to thousands of teacher resources, lesson plans, and rubrics.Logos Quiz Game Answers - Level 1 ArtoGraphedComputer Science amp Information Technology Armstrong Atlantic ...www.businessballs.comnbsp#8250#32human resourcesFree jobs interviews tough questions and answers, interviews tips and techniques to use or for practice plus free online training for management, sales, marketing ...www.footprintnetwork.orgenindex.phpGFNpagecalculatorsFootprint Calculator . How much land area does it take to support your lifestyle Take this quiz to find out your Ecological Footprint, discover your biggest areas of ...Technology Services The College of Saint Roseits.strose.eduWhere do I get technology support The offices that provide direct customer support are listed below. You can find individual staff contact information on the About ...Footprint Calculatorwebassign.netOnline homework and grading tools for instructors and students that reinforce student learning through practice and instant feedback.


text:
Today, Garrett Bauer left behind his life in New York to begin serving a 9 year prison sentence for insider trading. Bauer pled guilty last December for his role in a 3-man ring that used confidential information on publicly traded companies. While Bauer was the man who executed the trades, Matthew Kluger was the source of the information from the law firms where he worked. Kenneth Robinson was the middle man who passed the information between the source and the trade, allowing the scheme to go undetected for 17 years. Kluger, who also pled guilty reported to prison last week to begin his 12 year prison sentence, while Robinson will report to prison for his 27 month sentence in September. Robinson cooperated with authorities by providing information and recording phone calls which led to the arrests of both Bauer and Kluger.
Bauer checked in to the federal prison camp today in Montgomery, AL on the grounds of Maxwell Air Force Base. It has been just over 6 weeks since U.S. District Judge Katharine Hayden in New Jersey handed down two of the longest sentences for insider trading (Bauer's and Kluger's) in U.S. history. In addition, Bauer forfeited all of his assets, which authorities have estimated to be about $25 million. Convicted insider trader Raj Rajaratanam, hedge fund manager who went to trial last year and was found guilty on all charges, got 11 years.
Bauer's life has been on hold since his arrest in April 2011. To be productive and make amends for his illegal actions, he acknowledged his faults to not only the prosecutors in New Jersey, but to students around the country through campus visits and Skype presentations. No audience was too small as Bauer told students, "My hope is that you will never cross the line that I did." From his first presentation at the Manhattan Jewish Experience at the Fifth Avenue Synagogue last September, to his final presentation at Fairleigh Dickinson University in Teaneck, NJ just days before his prison sentence, Bauer was on a mission to right his wrong through sharing his story. However, Judge Hayden did not see it that way and the 147 presentations that Bauer conducted over 9 months did not help his cause, but it did make a difference in many peoples' lives. Hundreds of letters sent to the judge from faculty and students reflected the power of Bauer's speech.
The past 6 weeks were painful for Bauer. His bank accounts were seized upon his arrest and he subsequently relinquished his apartment to the government as part of his settlement with them. He had stopped paying his mortgage, property taxes and HOA fees because he had no money. It was an odd existence living in a 5,000 sq.ft. apartment in Manhattan but only living off the cash in his pocket. The only money he was allowed to keep, and live on, came from selling personal belongings and furniture, which he slowly sold off on Craigslist and Ebay.
Once his prison sentence was handed down on June 4, he began to accelerate the packing and sale of his belongings. By July 4th, the apartment was empty and his few remaining items from his life were boxed and stored in a basement of a friend's home on Long Island. "I look at all my stuff in boxes in that basement and think, 'This is all that's left of my life,'" he told me in an interview last week. "I find myself taping up boxes and thinking that when I undo this tape that my life will be so much different years from now." Bauer, who turned 45 last Saturday, spent the last few weeks living with friends in New York and then staying with family in Florida.
Bauer told me, "For the past 2 weeks I've eaten more barbecue ribs and ice cream than I have in my entire life." With regard to the emotional toll this has taken on him he said, "I've had my moments of crying but the more that I'm around people, the better I've been. I just am having a hard time believing that this is the way it turned out for me."
Bauer's older brother met him at Atlanta's Hartfield-Jackson airport on Monday to drive down I-85 South to Montgomery, AL. "I want to scope out the town so that I get a feel for where I am. I've never been to Alabama."
This morning I received a final text from Garrett Bauer, "Going over there now! I'm nervous, but not scared."
With good behavior, Garret Bauer will be released from federal prison in January 2020.


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Most people are familiar with polypro (ie polypropylene) rope from their hardware or home improvement store. The kind sold there is mostly mono filament polypro which is hard and brittle feeling and is usually offered with either 3 strands that are twisted together, such as the kind we offer here or flat-ish and hollow inside.
Did you know that there is another kind of polypro rope that's quite soft?This kind of rope is called Multi Filament Polypro and is used in many applications where it's expected that someone will come in contact with the rope. Because of it's soft feel it's used as barrier rope and swimming pool lane rope, to name two. We currently offer this MFP rope in our clearance section at rock bottom prices such as this 1/4″ Black and White rope for only $50.00 for 1000 ft!
Make sure to check our Clearance section often where we offer first quality products at deeply discounted prices.


text: Publications Search Results Users may wish to consult What's Included in the Bibliography. Results 1 - 1 Results per page: 25 50 100 Sort by: Pub Date (newest) Title Recent Additions Author Clear All Pub. Year Citation 2003 Latkin, C.A., Curry, A. Stressful neighborhoods and depression: A prospective study of the impact of neighborhood disorder. Journal of Health and Social Behavior. 44, (1), 34-44. Full Text Options: Worldcat Google Scholar Export Options: RIS EndNote XML [1]


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Pearl Jam performed in Kansas City on Monday night. Lead singer Eddie Vedder wore a Willie Wilson jersey during the show. The crowd started chanting Willie's name. ATH received this photo of the Royals Hall of Fame member presenting the jersey to Vedder.
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JOHNSONBURG – Johnsonburg Elementary School's newest first grade teacher is Chelsea Mulcahy.
Mulcahy is a 2005 graduate of St. Marys Area High School and upon completion of high school, Mulcahy attended Saint Bonaventure University in New York for her Bachelor of Science degree in Elementary Education. Mulcahy then continued for her Masters in Childhood Literacy, also at Saint Bonaventure University.
In addition to being a teacher, Mulcahy is actively involved at the St. Marys Community Pool and an Assistant Varsity Girls Soccer Coach at St. Marys Area High School.
Philip L. Woodford Jr., 52, of 8975 Hawthorne Rd., La Plata, Md., died August 23, 2011, as the result of a motorcycle accident, in White Plains, Md. He was born May 10, 1959, in St. Marys, the son of Philip L. and Lois Milliron Woodford Sr. On April 10, 1983, he married Anne' Bonnagio, who survives.
Mr. Woodford had been a La Plata resident for 10 years. He was a member of the Moose Club. He enjoyed golfing, bowling and coin collecting. He was an avid Steelers fan.
With some budget cuts being anticipated, officials at the North Central Pennsylvania Regional Planning and Development Commission [NCPRPDC] are looking the region's various CareerLink sites.
"As we're working through the process with our partners, to date we've confirmed that Potter, McKean and Elk [counties] will all be able to remain comprehensive centers," said Susie Snelick, Workforce Investment Board Director. "That is the result of partners stepping up and looking at cost-savings, looking at budgets and being able to continue to contribute and maintain comprehensive sites.
William L. Jacob, 94, of Silver Creek Terrace, St. Marys, died Thursday, August 25, 2011 at the Elk Regional Health Center.
He was born October 4, 1916, in St. Marys, son of the late A. Thomas and Catherine Kriegel Jacob. He married Rose McLeod and she preceded him in death on May 6, 1979. He then married Virginia Richards and she preceded him in death on January 2, 1995.
He was a lifelong resident of the area and was a graduate of Central Catholic High School, Class of 1934. He was a self-employed plasterer.
Ronald L. Kuppelwiser, 68, of Long View Court, Weedville, died Wednesday, August 24 at the Christ the King Manor in DuBois after a lengthy illness.
He was born June 9, 1943 in St. Marys, son of the late Simon Kuppelwiser who died in 1983 and Hazel Aunkst Kuppelwiser who died in 2005.
He had lived in Lorain, Ohio for years. He was a tractor trailer driver for many years, working for Jefferson Trucking, Inc. in Ohio.
The Ridgway boys golf team continued its strong play in the early season with a 41-stroke margin of victory, 214-255, over visiting St. Marys in action Thursday afternoon at Laurel Mill Golf Course.
Matt Aiello of Ridgway was the medalist for the day with a low score of 40 for the Elkers. Teammate Jesse Reynolds was second with a 41.
ST. MARYS – The Elk County Catholic Lady Crusader tennis team evened their record at 1-1 as they defeated the Bradford Lady Owls by a 9-0 score in a match played at Benzinger Park Tuesday afternoon.
Elk Catholic will play host to the Brockway Lady Rovers this afternoon at 3:30 p.m. at Benzinger Park.
"Today we opened our District 9 season with a win over a good, but young, Bradford squad," said ECCHS coach Pete Meier.
At first singles, ECC's Abby Meyer defeated Emily Hardy 6-0, 6-4.
At second singles, Loreal Housler of Elk Catholic beat Amina Sosic 6-1, 6-2.
Over the past several years, numerous proposals have been offered in the State Legislature to eliminate or significantly change the current law regarding expansion of Sunday hunting allowances. But any expansion of Sunday hunting, which now allows just fox and coyote hunting, has been soundly denounced by the members of the Pennsylvania Farm Bureau and Ernest Mattiuz, vice president of the Elk County Farm Bureau.
After a meeting last month revealed that the Women, Infants and Children [WIC] program out of the North Central Pennsylvania Regional Planning and Development Commission [NCPRPDC] may face a $92,000 budget cut for the new fiscal year, officials expressed concern Wednesday morning after the figures had a chance to sink in.
Homer C. Wolfe Jr., age 62, Powers Avenue, Johnsonburg, died at home, Wednesday, Aug. 24, 2011, after a short illness.
He was born Oct. 8, 1948, in Ridgway, son of Homer C. Helen M. (Fitch) Wolfe Sr., deceased.
He married Arlean M. (Roessler) in June of 2009, she survives in Berlin Center, Ohio.
A graduate of Johnsonburg High School, he served for three years as a Marine in Vietnam. He retired from the State Police Agency of Oklahoma state and in 2005 retired as a Sergeant from the Oklahoma Army National Guard. He worked locally as a driver for ATA for six years.


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Hold down Control (PC) or Command (Mac) key + mouseclick to select more than one option
|Avg. Dinner Entrée
|Greater than $25
|$18.01 - $25
|$12.01 - $18
|$7.01 - $12
|Less than $7
Posted by Marybess, Office Manager on 02/02/2012
Our Williamsburg-based company has been ordering lunch from Simple Cafe for quite a while now. Not only is the food delicious and consistently fresh, but the service is above and beyond fantastic. The owner is a lovely woman who goes to great lengths to keep her customers happy. She even delivers orders herself when the restaurant is too busy. I definitely recommend Simple Cafe to anyone looking for a worthwhile meal from caring people.
Posted by Bill on 04/09/2011
I went here this morning with my girlfriend for brunch because our normal places were packed and Simple was nearly empty. I had a faint recollection of being there before and not being satisfied but at my gf's insistence went anyway. It took too long to get menus, I had to send my fresh squeezed orange juice back because there were nearly a dozen seeds floating at the top and when it returned I found out it was also very sour. My gf had to send her poached eggs back because they were extremely undercooked and my French Toast was tiny, bland, and they forgot to bring the syrup. It's a shame because the place is in a nice location and very pretty inside. PS the coffee was also disappointing
Posted by williamsburgmom on 03/09/2011
My family of three came to check out this local restaurant with a certificate. The owner waited on us and apparently did NOT want to serve the last of the 11 of these certificates still circulating. It was our first time at the place. We ordered a simple meal, hamburger, steak,crab cakes. The food was fine, actually tasty enough, although it looked like I was given a tiny piece of scrap meat (as steak) but it was well seasoned and slathered in onions to hide the size and quality of the meat. It was the service that was atrocious. When we arrived there was one customer in the place that had finished eating and was surfing the net. We were greeted, and an order was placed. We were given a bottle of water and our food. No other service followed. Anything else we needed we had to get ourselves, including the bill. When we were given our bill insted of beaing charged 18% gratuity we were charged $18 tip on a $30 something bill. When we asked the owner to correct it, she got so ANGRY about the coupons. We were never given a chance. When I told her the service was poor, but the food fine, she couldn't believe I thought so. As if we weren't there. She did correct the bill grudgingly but basically told us she didn't want us to return. And we won't.
Posted by cam on 09/20/2010
I recently moved to the neighborhood and tried simple for brunch last weekend and was super pleased with my decision. My friend and I sat in a cozy corner and sipped yummy peach mimosas while we leisurely ate the brunch buffet. Everything was delicious from the quinoa salad to the potatoes to the little homemade donuts! They really thought of it all. The service was warm just like the atmosphere. I would highly recommend simple and will be going back next weekend!
Posted by anonymous on 07/20/2010
My girlfriend and I have passed by this placed several times, but never bothered to go in. We bought a gift certificate to this place online, thinking it was a different restaurant. The food was subpar, I had the Eggs Norwegian and my lady had the Crouque Madam, mine was way too wet, her's was way to dry. The potatoes that came on the side of mine were a strange, kind of mushy texture. The service was extremely slow. They weren't too pleased that we had the gift certificates, but if they don't want people to use them, they shouldn't sell them. The apple tart wasn't too shabby. Don't waste your time or your money. It seemed like amateur hour in there to me.
Posted by phg on 02/03/2010
Nice staff, space is cute but unfortunately the food does not follow the rest.
Posted by ben on 10/22/2009
i went here looking for good sandwiches, and a cute spot and thats exactly what i got. friendly service, albeit a little slow, from a nice french waitress. they bake their own bread for their sandwiches which is so good. value is perfect. just a great spot.


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Monday, October 3, 2011
Gonna Pay For This Tomorrow
Really Tasty Sandwich
by ScentedNectar
I forgot to mention that there is also, what I think are shiitake mushrooms, and also, there was a long piece of cucumber that I took out because I don't like it in these.
12 comments:
Syncronicity, how does it work? I now see there is a sandwich blogpost over at Laden's blog today too from 12 hours ago. Weird. :)ReplyDelete
Not only is this a post about a sandwich, but it is also a post about an elongated sandwich.ReplyDelete
I wasn't at all thinking of elongated things when I bought it, although Freudians might argue otherwise. :)ReplyDelete
Even though not vegetarian, I love these vegetarian sandwiches - you gotta admit they come up with some amazing and tasty stuff.ReplyDelete
They also have some really great vegetarian and vegan sweets.
Some vegan cookbooks are amazing.
This place a lot of them that do have meat too. A ton of them, in fact. I'm a vegetarian but not for the woo purposes I believed in when I first became one. I'm just a little grossed out aesthetically now by slabs of muscle. I get enough animal foods though for a good balance (I hope) by eating dairy, eggs, and a tiny bit of seafood that sneaks into some things I eat like Caesar salad dressing.ReplyDelete
I had no idea that Vietnamese food was so good, until I tried out this take-out place. If you have one near you, I highly recommend it. The sandwiches are called 'banh mi' I think. Yum. :)
Ah, good news to report. My somewhat fast metabolism has done it's thing, and it barely burned at all. Nothing at all like last time I had hot pepper on these sandwiches. Whew! :)ReplyDelete
That's one thing that sucks about Europe, we don't have "ethnic restaurants".ReplyDelete
The USA is the only place world that has that much variety in every city. You guys are lucky, you can eat a different meal each meal of every day and never try everything lol.
I had no idea. Sounds like Europe is a ripe market to open some restaurants up, in that case. I wonder why no one's exploiting it. I always thought it was pretty much the same there. Here in Toronto, we have specialty ones from almost every other country. Even to order in on delivery in the same manner as ordering pizza, there's quite a variety.ReplyDelete
Vietnamese rolls are everywhere here in the western suburbs.ReplyDelete
Very popular.
A result of Australia "going all the way with LBJ" in his brutally illegal war against both the North *and* the South Vietnamese. (And then the Laotians, the Cambodians, etc, as he was a mission creep).
Australia once tried to invade Vietnam on behalf of the Yanks, but failed. I am surprised that enormous numbers of Vietnamese refugees here, and their offspring, bear so little malice against we illegal and brutal invaders.
(I just missed, by the skin of my teeth, getting "called up" for duty in the Australian Army in Vietnam. I was an Army cadet at the time that Prime Minister Whitlam ended the draught.)
Their food is amazing, as you have discovered.
The irony is that the chilli originated in Central America, and as a result of wars, decimation, invasion by Europeans, the South East Asians now include it as a staple of their cuisine!
Good thing you missed having to fight. Being lawfully ordered to kill would suck. I'd have to be one of those conscientious objectors and given an office job if drafted.ReplyDelete
Back when PZ wrote an article about how his son was off to join the military, I noticed he made no mention of the ethics, just a nice little family pic with the kid in uniform, off to kill, kill, kill. I wonder if army guy over on FreeFromThoughts is his son.
At least wars meant that they got chili to add to their cuisine in Asia. And it sure does go well with it. :)
I do believe western europe does have some variety (like the UK and maybe germany), but even then, they don't come even close to the USA and Canada.ReplyDelete
I think the reason these restaurants haven't sprung up is because we don't have immigration here as much, which is why there aren't many ethnic restaurants.
If I ever have visitors from Europe, I'm going to take them to a different exotic restaurant every day.ReplyDelete
Just in delivery flyers alone, I have menus for Greek, Thai, Japanese, and some more that I'm forgetting.
UK for sure has Indian food, but that's probably just because they once 'colonized' India.


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The links below take you to dynamically generated lists of Duke of Abruzzi's peakbagging activity.
Reports showing a climber's buddies and other climbers. Click for More Info
Lists personally created by Duke of Abruzzi.
These reports show number of peaks climbed, highest point reached, and other statistics grouped by year or month.


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Swedish illustrator Mattias Adolfsson blends machines and animals with an elegant whimsy.
From the monthly archives:
July 2009
A Twitter rant/essay celebrating amateur instincts.
Writing for the Telegraph, Andrew Keen observes the new digital talent agency Polyphonic.
He concludes that "we might—just might—be on the verge of a glorious new age of the artist."
You may remember Keen from Cult of the Amateur, a book that largely asks you to appreciate his taste in culture.
Clearly this continues to frame his reportage. Somehow, the internet didn't make this "the age of the artist."
Providing ubiquitous and cheap tools to make, share, and consume media wasn't THE factor.
(Then everybody gets to participate. And why would we want that?)
No, what will give rise to the "glorious age" are outfits that can market and monetize artists.
People, like Keen, who can separate the wheat from the chaff. Tastemakers who can explain the virtues of "good" to us.
Note to Keen: In art (and all endeavors, including the technology you use), the "glorious age" is when anyone can participate.
It IS the amateur ethic—the lack of experience, the awkward groping to create—that produces something fresh.
Any painting, song, or film you've appreciated started with an "amateur." Pollock was an amateur. So was Yorke. Soderbergh too.
The very culture you disdain is the very culture that created this glorious age.
That artists can get support from middlemen like Polyphonic is helpful. Artists shouldn't starve.
But middlemen reframe things—eerily like reporters—and over time exert their own tastes on what they support.
This is what led us to the "inglorious age" that the internet thankfully disabled.
So, thanks for all the fish. But we've got our own boats now. We'll take it from here.
We occasionally use Twitter for rants or essays:
Publishers are the new Flickr Groups
Insanely adorable protagonist/antagonist portraits by Scott Campbell for Gallery 1988′s Crazy for Cult exhibit.
Thanks for getting the kids this far. We'll miss you.
A look at upgrades to the artist shop and the new artist dashboard. (Toggle the full screen icon on the player for best effect.)
Story art by Victoria Usova, a Ukranian-born New York-based fairy tale maker. Visit her portfolio and ETSY shop.
Click here to see the previous sneak peeks and other artist resources.
Lately I've rekindled a fascination with endpapers – the paper pasted to the inside cover that holds the book together. I've always felt they were a 'secret' additional source of information, whether it was the map of the island/world the book was exploring, or silhouettes of Nancy Drew solving various mysteries. Every day I'm inundated with massive doses of digital text that I quickly skim and then move on – endpapers are just the opposite, they invite me to stop and study their content and its relevance to the book, making reading that book much more of a therapeutic leisure activity than an information-gathering exercise. Both are valid activities, but oh, so different
Here's one great resource for endpapers: Drawger.com
Erwin Madrid is magic.


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Oakley Boulevard
Another UIC Student Robbed Near Campus
Another student was robbed this week near the University of Illinois at Chicago campus, prompting campus police to issue an alert.
UIC Student Robbed Blocks From Campus
A University of Illinois at Chicago student was robbed at gunpoint several blocks west of the main campus this week.
7 Dead, 24 Others Wounded In Weekend Violence
It has been a terrifying and deadly weekend in Chicago, with at least seven people dead and at least 24 more wounded in shootings since Friday afternoon.


text: Prev | Next Purchase this photo Bishop Nash/For The Herald-Dispatch Huntington's JR Howard (23) and Woodrow Wilson's DaiDai Fortune (32) battle for the ball on Friday, Dec. 20, 2013, at Huntington High School. Gallery: Huntington High vs. Woodrow Wilson, basketball Dec. 20, 2013 @ 10:21 PM Bishop Nash The Huntington Highlanders defeated the Woodrow Wilson Flying Eagles, 77-68, Friday night in Huntington. ()