Method for imaging and differential analysis of cells
First Claim
Patent Images
1. A method for classifying a specific cell as one of the following four types of cells, a viable cell, a necrotic cell, an early apoptotic cell in which a cellular membrane of the cell is still intact, and a late apoptotic cell in which the cellular membrane of the cell is not intact, using only a single nuclear marker and image data from the cell, the method comprising the steps of:
- (a) exposing the specific cell to only a single nuclear marker that will bind to DNA in a nucleus of the specific cell in the event that the cellular membrane of the specific cell is not intact;
(b) collecting image data from the specific cell, the image data comprising;
(i) a darkfield image of the specific cell, the darkfield image corresponding to a side scatter image of the specific cell;
(ii) a brightfield image of the specific cell; and
(iii) a fluorescent image of the specific cell, the fluorescent image enabling the location of the single nuclear marker in the specific cell to be determined;
(c) using the brightfield image of the specific cell and the fluorescent image of the specific cell to calculate a cytoplasmic size of the specific cell;
(d) analyzing the fluorescent image of the specific cell to determine if the nuclear marker is present in the nucleus of the specific cell;
(e) using the darkfield image of the specific cell to calculate a spatial scatter frequency metric indicative of a granularity of the specific cell; and
(f) classifying the specific cell as one of the four types of cells based on the following;
(i) the cytoplasmic size of the specific cell;
(ii) the darkfield scatter frequency metric of the specific cell; and
(iii) the presence of the nuclear marker in the nucleus of the specific cell.
3 Assignments
0 Petitions
Accused Products
Abstract
Provided are methods for determining and analyzing photometric and morphogenic features of small objects, such as cells to, for example, identify different cell states. In particularly, methods are provided for identifying apoptotic cells, and for distinguishing between cells undergoing apoptosis versus necrosis.
67 Citations
20 Claims
-
1. A method for classifying a specific cell as one of the following four types of cells, a viable cell, a necrotic cell, an early apoptotic cell in which a cellular membrane of the cell is still intact, and a late apoptotic cell in which the cellular membrane of the cell is not intact, using only a single nuclear marker and image data from the cell, the method comprising the steps of:
-
(a) exposing the specific cell to only a single nuclear marker that will bind to DNA in a nucleus of the specific cell in the event that the cellular membrane of the specific cell is not intact; (b) collecting image data from the specific cell, the image data comprising; (i) a darkfield image of the specific cell, the darkfield image corresponding to a side scatter image of the specific cell; (ii) a brightfield image of the specific cell; and (iii) a fluorescent image of the specific cell, the fluorescent image enabling the location of the single nuclear marker in the specific cell to be determined; (c) using the brightfield image of the specific cell and the fluorescent image of the specific cell to calculate a cytoplasmic size of the specific cell; (d) analyzing the fluorescent image of the specific cell to determine if the nuclear marker is present in the nucleus of the specific cell; (e) using the darkfield image of the specific cell to calculate a spatial scatter frequency metric indicative of a granularity of the specific cell; and (f) classifying the specific cell as one of the four types of cells based on the following; (i) the cytoplasmic size of the specific cell; (ii) the darkfield scatter frequency metric of the specific cell; and (iii) the presence of the nuclear marker in the nucleus of the specific cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
-
-
10. A method for classifying a specific cell as one of the following four types of cells, a viable cell, a necrotic cell, an early apoptotic cell in which a cellular membrane of the cell is still intact, and a late apoptotic cell in which the cellular membrane of the cell is not intact, using only a single nuclear marker and image data from the cell, the method comprising the steps of:
-
(a) exposing the specific cell to only a single nuclear marker that will bind to DNA in a nucleus of the specific cell in the event that the cellular membrane of the specific cell is not intact; (b) collecting image data from the specific cell, the image data comprising; (i) a darkfield image of the specific cell, the darkfield image corresponding to a side scatter image of the specific cell; and (ii) a fluorescent image of the specific cell, the fluorescent image enabling the location of the single nuclear marker in the specific cell to be determined; (c) analyzing the fluorescent image of the specific cell to determine if the nuclear marker is present in the nucleus of the specific cell; (d) using the darkfield image of the specific cell to calculate a spatial scatter frequency metric indicative of a granularity of the specific cell; (e) classifying the specific cell as being a viable cell when; (i) the nuclear marker is not present in the nucleus of the specific cell; and (ii) the darkfield scatter frequency metric of the specific cell is smaller than a darkfield scatter frequency metric of a cell known to be an early apoptotic cell in which a cellular membrane of the cell is still intact; (f) classifying the specific cell as being an early apoptotic cell when; (i) the nuclear marker is not present in the nucleus of the specific cell; and (ii) the darkfield scatter frequency metric of the specific cell is larger than a darkfield scatter frequency metric of a cell known to be a viable cell; (g) classifying the specific cell as being a late apoptotic cell when; (i) the nuclear marker is present in the nucleus of the specific cell; and (ii) the darkfield scatter frequency metric of the specific cell is larger than a darkfield scatter frequency metric of a cell known to be a necrotic cell; and (h) classifying the specific cell as being a necrotic cell when; (i) the nuclear marker is present in the nucleus of the specific cell; and (ii) the darkfield scatter frequency metric of the specific cell is smaller than a darkfield scatter frequency metric of a cell known to be a late apoptotic cell. - View Dependent Claims (11)
-
-
12. A method for classifying a population of biological cells into four different types of cells, the four types of cells consisting of viable cells, necrotic cells, early apoptotic cells in which a cellular membrane of the cell is still intact, and late apoptotic cells in which the cellular membrane of the cell is not intact, using only a single nuclear marker and image data acquired from each cell in the population of cells, the method comprising the steps of:
-
(a) exposing each cell in the population of cells to only one nuclear marker that will bind to DNA in a nucleus of the cell in the event that the cellular membrane of the cell is not intact; (b) collecting image data from each cell in the population of cells, the image data for each cell comprising; (i) a darkfield image of the cell, the darkfield image corresponding to a side scatter image of the cell; (ii) a brightfield image of the cell; (iii) a fluorescent image of the cell, the fluorescent image enabling the location of the nuclear marker in the cell to be determined; (c) for each specific cell in the population of cells, using the brightfield image of the specific cell and the fluorescent image of the specific cell to calculate a cytoplasmic size of the specific cell; (d) for each specific cell in the population of cells, using the darkfield image of the specific cell to calculate a spatial scatter frequency metric indicative of a granularity of the specific cell; (e) generating a dot plot where data from each cell in the population of cells is displayed, the dot plot being generated using the cytoplasmic size of each cell as a first axis of the dot plot, and the spatial scatter frequency metric of the darkfield image of each cell as a second axis of the dot plot; and (f) using the dot plot to classify the population of cells into the four different types of cells based on the relative location of each cell in the dot. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20)
-
Specification