News Column

Patent Issued for Automated Analysis of Images Using Bright Field Microscopy

June 19, 2014



By a News Reporter-Staff News Editor at Computer Weekly News -- Institute for Systems Biology (Seattle, WA) has been issued patent number 8744164, according to news reporting originating out of Alexandria, Virginia, by VerticalNews editors.

The patent's inventors are Ozinsky, Adrian (Seattle, WA); Selinummi, Jyrki Juhani (Nokia, FI); Shmulevich, Ilya (Seattle, WA); Ruusuvuori, Pekka (Pori, FI).

This patent was filed on April 4, 2011 and was published online on June 3, 2014.

From the background information supplied by the inventors, news correspondents obtained the following quote: "Cell detection is a fundamental procedure in any biomedical study where microscopy images of cell populations are used. Cell detection can be used for counting the individual cells, or as a basis for further analysis, ranging from feature extraction to single cell tracking. This procedure has been intensively studied in the image processing community.

"Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.

"The development of highly specific stains and probes, for example the green fluorescent protein and its derivatives, have made fluorescence microscopy the standard tool for visualization and analysis of cellular functions and phenomena. On the other hand, automated microscopes and advances in digital image analysis have enabled high-throughput studies automating the imaging procedure and cell based measurements. In fluorescence microscopy of eukaryotic cells, automated single-cell quantification can be achieved using multiple fluorescent probes and channels in a single experiment. The first fluorescence channel enables detection of stained nuclei, resulting in markers for cell locations. The second fluorescent channel visualizes the areas occupied by whole cells or cytoplasm, for example by a cytoskeletal actin stain, as described in Moffat J, Grueneberg D A, Yang X, Kim S Y, Kloepfer A M, et al. (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124: 1283-1298. Alternatively, a nonspecific subcellular stain can be used for whole cell detection. Regardless of the approach for whole cell staining, cells that are touching or partly overlapping can be automatically separated with the help of the nuclei markers of the first channel, as describe in Carpenter A E, Jones T R, Lamprecht M R, Clarke C, Kang I H, et al. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7: R100. Finally, subcellular phenomena are quantified by measuring different properties of the first and second channels, or by using additional organelle and molecule specific probes and extra fluorescence channels, for example in colocalization measurements, as described in Bolte S, Cordelieres F P (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224: 213-232.

"Because of the limited number of fluorescent channels available, and because of partly overlapping excitation and emission spectra of the probes, studies involving subcellular colocalization are commonly carried out without nuclear or whole cell staining. As a consequence, cell-by-cell measurements are not possible. Single cell measurements are also difficult or even impossible in cells that are used for negative control, where the lack of fluorescence is used for the detection of some phenomena. Furthermore, there are other limitations in fluorescence microscopy, such as phototoxicity and imaging setup complexity. These problems have motivated the search for alternate methods to replace at least some of the fluorescence channels with standard transmitted light microscopy.

"A number of problems in counting cells using the conventional methods have been observed. These problems include the contamination of the cells (and possibly the growth culture in which they are found) with extraneous chemical substances that are needed for counting, but that make the further use of the cells impractical or impossible.

"There is a need for systems and methods that provide the ability to count cells without subjecting the cells to extraneous chemicals."

Supplementing the background information on this patent, VerticalNews reporters also obtained the inventors' summary information for this patent: "While the invention will be described using a microscope slide having a surface upon which cells to be examined are situated, it is to be understood that the invention can equally well be practiced using other well known substrates for supporting cells, such as plates, objects having wells defined therein, culture dishes, cell growth media, and their equivalents.

"In one aspect, the invention features a method of automatically identifying the number of cells present in a sample. The method comprises the steps of providing an optically transparent supporting surface, the optically transparent supporting surface situated for observation in an optical microscope having a sensor sensitive to optical illumination, the sensor having an output terminal configured to provide as output a signal representative of a field of view monitored by the sensor; providing a sample comprising at least one cell situated on the optically transparent supporting surface; deliberately operating the optical microscope in bright field mode with optical illumination so as to focus at one or more different focal planes situated along a direction normal to the optically transparent supporting surface, so that the at least one cell is within the field of view of the sensor; observing with the sensor an image selected from the group of images consisting of one or more bright spots and one or more dark spots, the image corresponding to a specific focal condition; providing from the output terminal of the sensor an output signal representative of the image; processing the output signal representative of the image to compute a number of bright spots or a number of dark spots; and reporting the number of bright spots or the number of dark spots as the number of cells present in the sample.

"In some embodiments, the sample comprising at least one cell is free of a staining agent. In some embodiments, the sample comprising at least one cell is free of a fluorescent agent. In some embodiments, the method of automatically identifying the number of cells present in a sample further comprises the step of having a human operator view an image observed by the sensor.

"In some embodiments, the step of processing the output signal representative of the image is performed in a computer-based analyzer. In some embodiments, the computer based-analyzer provides a synthetic image of the sample comprising at least one cell, the synthetic image including an outline of the at least one cell in false color. In some embodiments, the method of automatically identifying the number of cells present in a sample further comprises the step of focusing on the at least one cell, the step of focusing on the at least one cell being performed prior to the step of observing with the sensor an image selected from the group of images consisting of one or more bright spots and one or more dark spots.

"In some embodiments, the specific focal condition is an out-of-focus condition.

"In another aspect, the invention relates to an automated image processing system. The system comprises an optical microscope having a sensor sensitive to optical illumination, the sensor having an output terminal configured to provide as output a signal representative of a field of view monitored by the sensor, the optical microscope configured to allow operation of the optical microscope in bright field mode with optical illumination and configured to allow the optical microscope to change focus along a direction normal to an optically transparent supporting surface situated for observation in the optical microscope so as deliberately to attain at least one image along the direction normal to the optically transparent supporting surface of a sample situated within the field of view of the optical microscope; a computer-based image processor configured to receive the output signal representative of a field of view monitored by the sensor from the sensor, the image processor configured to identify one or more images out of the at least one image, the image processor configured to analyze the at least one image to deduce a property of the sample from the one or more images; and a reporting apparatus in communication with the computer-based image processor, the reporting apparatus configured to provide a report of the property of the sample.

"In some embodiments, the at least one image is an image selected from the group of images consisting of one or more bright spots and one or more dark spots; the image processor is configured to compute a number of bright spots or a number of dark spots in the image; and the property reported by the reporting apparatus is the number of bright spots or the number of dark spots, so that the property reported is a number of cells present in a sample situated on a portion of the optically transparent supporting surface that is situated within the field of view of the optical microscope. In some embodiments, the automated image processing system further comprises an actuator configured to change a focal condition of the optical microscope. In some embodiments, the automated image processing system further comprises a computer-based control apparatus configured to control the focal condition of the optical microscope by driving the actuator.

"In some embodiments, the computer-based control apparatus configured to control the focal condition of the optical microscope is configured to operate to focus at one or more different focal planes situated along the normal to the optically transparent supporting surface on one or more cells in the field of view. In some embodiments, the automated image processing system further comprises an actuator configured to change a lens so as to change a magnification or a dimension of a field of view of the optical microscope.

"In some embodiments, the reporting apparatus provides a synthetic image. In some embodiments, the synthetic image includes false color. In some embodiments, the reporting apparatus provides a report that is recorded for later use. In some embodiments, the reporting apparatus provides a report that is displayed to a user. In some embodiments, the optical microscope is configured to allow simultaneous mounting of the sensor and an eyepiece suitable for a human operator. In some embodiments, the automated image processing system further comprises one or more power supplies to operate the optical microscope, the computer-based image processor, and the reporting apparatus.

"In some embodiments, the at least one image is selected from a bright field z-stack of images along a z-dimension; the image processor is configured to compute a variation with respect to the z-dimension of the intensity values within the x,y plane between a first image and a second image of the bright field z-stack, to construct a two-dimensional projection image of increased contrast, and to deduce from the two-dimensional projection image of increased contrast a feature of at least one cell of the sample; and the reporting apparatus is configured to report the feature of the at least one cell present in the sample. In some embodiments, the property is a border of the at least one cell. In some embodiments, the system is configured to spatially distinguish at least one cell from another cell within the sample

"In yet another aspect, the invention features a method of automatically identifying a feature of a cell present in a sample. The method comprises the steps of: providing an optically transparent supporting surface, the optically transparent supporting surface situated for observation in an optical microscope having a sensor sensitive to optical illumination, the sensor having an output terminal configured to provide as output a signal representative of a field of view monitored by the sensor; providing a sample comprising at least one cell situated on the optically transparent supporting surface; deliberately operating the optical microscope in bright field mode with optical illumination so as to focus at one or more different focal planes situated along a direction normal to the optically transparent supporting surface to form a bright field z-stack of images along a z-dimension, so that the at least one cell is within the field of view of the sensor; observing with the sensor a plurality of images selected from the bright field z-stack; providing from the output terminal of the sensor an output signal representative of the plurality of images; processing for at least two of the plurality of images the output signal representative of the plurality of images to obtain intensity values of pixels within an x,y plane; measuring a variation with respect to the z-dimension of the intensity values within the x,y plane between a first image and a second image of the at least two of the plurality of images; constructing a two-dimensional projection image of increased contrast; deducing from the two-dimensional projection image of increased contrast a feature of the at least one cell; and reporting the feature of the at least one cell present in the sample.

"In some embodiments, the feature is an observable property of the at least one cell. In some embodiments, the observable property is a border of the at least one cell. In some embodiments, the at least one cell is spatially distinguished from another cell within the sample.

"In some embodiments, images are acquired at several focus levels forming a bright field z-stack, and by measuring the intensity variations of this stack over the z-dimension, a new two dimensional projection image of increased contrast is constructed and analyzed. In one embodiment, with additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. In another embodiment, no staining is required.

"The foregoing and other objects, aspects, features, and advantages of the invention will become more apparent from the following description and from the claims."

For the URL and additional information on this patent, see: Ozinsky, Adrian; Selinummi, Jyrki Juhani; Shmulevich, Ilya; Ruusuvuori, Pekka. Automated Analysis of Images Using Bright Field Microscopy. U.S. Patent Number 8744164, filed April 4, 2011, and published online on June 3, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8744164.PN.&OS=PN/8744164RS=PN/8744164

Keywords for this news article include: Institute for Systems Biology.

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