Methods and apparatus for measuring multiple optical properties of biological specimens
First Claim
1. A method for generating cell constituent data that characterizes a population of cells, the method comprising:
- (a) scanning the cell population with a laser beam to produce digital data samples, different said samples representing different locations within the cell population;
(b) storing said digital data;
(c) locating a cell within said population;
(d) defining a neighborhood of digital data samples that includes the digital data samples corresponding to the located cell;
(e) estimating a background level for each digital data sample of the neighborhood based upon stored digital data from corresponding scan locations in prior or subsequent neighborhoods;
(f) correcting each of said digital data samples corresponding to the neighborhood with the estimated background level; and
(g) summing said corrected digital data samples in the neighborhood to generate the cell constituent data.
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Abstract
The invention relates to an apparatus and methods for generating multiparameter optical data that characterize a population of cells. The invention includes the steps of scanning the cell population with a beam to produce sets of digital data samples, each sample set of digital data representing multiparameter optical interactions from a specific location within the cell population; storing the digital data, e.g., in a computer memory; locating a cell within the population, e.g., by comparing the digital data to a preselected threshold value; defining a neighborhood around the digital data representing the located cell; estimating a background level for the neighborhood based upon digital data corresponding to locations outside the neighborhood; and correcting each of the samples corresponding to the neighborhood with the estimated neighborhood background level to generate the optical data. The invention further relates to specific methods of background correction and data calibration as well as specific sampling features to enable precise estimates of multiple cellular constituents or other cell properties at high rates of speed.
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Citations
38 Claims
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1. A method for generating cell constituent data that characterizes a population of cells, the method comprising:
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(a) scanning the cell population with a laser beam to produce digital data samples, different said samples representing different locations within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood of digital data samples that includes the digital data samples corresponding to the located cell; (e) estimating a background level for each digital data sample of the neighborhood based upon stored digital data from corresponding scan locations in prior or subsequent neighborhoods; (f) correcting each of said digital data samples corresponding to the neighborhood with the estimated background level; and (g) summing said corrected digital data samples in the neighborhood to generate the cell constituent data. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. An apparatus for generating cell constituent data that characterizes a population of cells comprising:
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(a) a scanner for scanning the cell population with a laser beam and a motorized stage to move the cell population at a right angle to the laser scan to produce digital data samples from light emitted from the cells upon laser excitation, different said samples representing different locations along a scan line within the cell population; (b) a memory to store said digital data; (c) means for locating digital data samples in memory corresponding to a cell within said population; (d) means for defining a neighborhood around the located cell; (e) means for estimating a background level for each digital data sample of the neighborhood based upon stored digital data corresponding to scan locations in prior or subsequent neighborhoods; and (f) means for correcting each of said digital data samples corresponding to the neighborhood with the estimated neighborhood background level to generate the cell constituent data.
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17. A method for characterizing a population of cells, the method comprising:
(a) scanning the cell population on a surface along a scan line with a laser beam with a spot size comparable in size to the cells to be scanned to produce analog signals from a sensor that detects light emitted by the cells and sampling said analog signals to produce digital data samples, said samples being taken at a sampling rate set such that the distance travelled per time as the spot is scanned between consecutive sampled locations approximates said spot size;
(b) displacing the scan line of said laser with respect to said surface such that different said samples represent different locations within the cell population; and
(c) processing the digital data to generate cell constituent data that characterizes the cells in the population.- View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. An apparatus for measuring and displaying cellular properties of a population of cells comprising:
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a laser source for generating a beam of light to illuminate the cells to be measured with a beam spot comparable in size to the cells to be scanned, an optical signal being emitted as a result of said spot illuminating said cells; a surface upon which the cells are located; an optical path for directing said spot from said beam source to the cells on said surface; a scanner interposed in said optical path between said beam source and the cells for scanning aid spot across said surface along a scan line; a sensor for measuring said optical signal; an analog-to-digital converter arranged to sample said optical signal at a specified rate and to produce digital data;
whereby said sampling rate is set such that the distance traveled per time as the spot is scanned between consecutive sampled locations approximates said spot size;a means for displacing the scan line of said laser with respect to said surface such that said laser spot scans continuously over a plurality of cells on said surface; a data processor for deriving cell constituent data from said digital data corresponding to individual cells, and processing said cell constituent data to display cellular properties of the cell population; and a memory for storing said digital data produced by said converter. - View Dependent Claims (36, 37, 38)
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Specification