Image analysis – Applications – Biomedical applications
Reexamination Certificate
1998-04-24
2002-10-01
Patel, Jayanti K. (Department: 2723)
Image analysis
Applications
Biomedical applications
C435S029000, C435S040500, C436S172000
Reexamination Certificate
active
06459805
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a system for quantifying relative cell numbers in tissue culture containers, and more particularly to a system for quantifying relative cell numbers in tissue culture containers using fluorescence digital imaging microscopy.
2. Description of the Prior Art
Measurement of relative cell numbers (total and/or viable only) is necessary for a wide variety of biological, immunological, and therapeutic studies and often requires analyzing many replicate samples. The use of multi-well tissue culture plates, such as the 96-well plates, provides a convenient format for such assays. Microwell assays for relative cell number have used radioactive isotopes (
51
Cr release or
3
H thymidine incorporation), colorimetric substrates produced by viable cells (MTT), or fluorescent dyes that accumulate in viable cells (fluorescein diacetate) or all cells (Hoechst 33342, ethidium bromide, etc.). Assays using radioactive isotopes require specialized waste disposal and the risk of exposure to radioactivity, while calorimetric assays may provide less precision or dynamic range than isotopic assays. Assay systems using fluorescent dyes are particularly attractive because of their ease of use, lack of radioactive waste, short incubation times, and because of the ability to rapidly measure cell numbers directly without disrupting cells.
Measuring the fluorescence of a sample involves illuminating the sample with light of suitable wavelengths, and recording the intensity of the fluorescence produced by the sample. Fluorescence readers that are currently commercially available use a photomultiplier tube to directly measure total fluorescence in a well, without using a focusing mechanism to measure the fluorescence of defined areas of a well. Since photomultiplier tubes detect total fluorescence, they cannot discriminate between background fluorescence and intracellular fluorescence. Consequently, assays must rely on rinsing steps to eliminate background fluorescence.
Furthermore, existing fluorescence readers have limited flexibility. For example, the Baxter Pandex FCA required the use of custom Pandex plates to wash the cells and an internal bead standard using custom Fluoricon reference beads. Therefore, existing florescence readers offer poor flexibility, low sensitivity and dynamic range, especially when background fluorescence is high.
Another limitation of commercial fluorescence readers and existing methods for quantification of cell number is the inability to discriminate between the fluorescence from the viable cells and background fluorescence in the wells from dye in the medium and dead cells.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a high degree of sensitivity and dynamic range for measurements of the number of viable cells.
It is a further object of the invention to provide a high degree of sensitivity and dynamic range for viable cell number measurements in the presence of large numbers of non-viable cells.
It is a further object of the invention to create a system for quantifying cellular fluorescence in situ, using a variety of tissue culture plate formats.
In response to these goals, a computer-controlled digital imaging microscopy scanning system is developed as described herein. The system is configured to accommodate a variety of tissue culture plates. Focusing optics are used to form an image of a defined area of the plate, which image is recorded by a recording device and digitized. The digital image, in the form of fluorescence light intensity at each pixel of the image, is then manipulated by the computer software to reduce background noise and extract desired information. The plate is mounted on a motorized stage, the movement of which is controlled by the computer. As the stage moves according to the computer commands, a series of images, or frames, are recorded, the frames covering the wells to be measured. This is referred to as a scan. During such a scan, the movement of the stage, the recording and digitizing of the images and the extraction of information occur in a synchronized manner under computer control.
The stage is designed to accommodate tissue culture plates of various formats, and the computer software is designed to control the stage movement for scanning the plate according to the format inputted to the computer. The algorithm for automatic determination of the scanning movement is described in more detail hereinafter.
The software quantifies fluorescence for the sample according to a predetermined method, described in more detail hereinafter, and sends the results both to the computer screen and to a data file. The software allows for two types of calibration, and provides easy to use menus for entering configuration, calibration and assay parameters, or data analysis specifications.
For the purpose of relative cell number determination, total fluorescence of a well is obtained by summing the fluorescence light intensities of all pixels in all frames for the well. The software provides for thresholding ability to enhance signal and reduce noise in the digital images. For example, fluorescence light intensities that are below a predetermined threshold may be rejected by using a Look Up Table. Because intracellular fluorescence is concentrated in a small area and is more intense, while background fluorescence is much more diffuse and is less intense, thresholding effectively reduces the background noise and increases the dynamic range of the cell number measurement. For samples with high background fluorescence, this technique accomplishes cell number measurement in one step, and eliminates the need for extra steps, such as rinsing, which would be otherwise required to reduce background fluorescence.
A method is also described for enhancing the dynamic range and accuracy of viable cell number measurements by treating the sample with a second dye to quench background fluorescence from the medium and non-viable cells. The first (fluorescent) dye accumulates in viable cells, while the second (quenching) dye enters non-viable cells but not viable cells, thereby quenching the fluorescence of the first dye in non-viable cells and the medium. When fluorescein diacetate (FDA) is used as the fluorescent dye, eosin Y (2′4′5′6′-Tetrabromofluorescein) may be used to quench the background fluorescence. When calcein-AM (Glycine, N,N′-[[3′,6′-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthene]-2′,7′-diyl]bis(methylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis(acetyloxy)methyl] ester) (Molecular Probes, Inc., Eugene, Oreg.) is used as the fluorescent dye, trypan blue (3,3′-[3,3′-Dimnethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalene-disulf acid] tetrasodium salt) may be used to quench the background fluorescence.
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Frgala Tomas
Reynolds C. Patrick
Children's Hospital Los Angeles
Hogan & Hartson LLP
Patel Jayanti K.
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