Patent US4765737A successfully refreshed from Google Patents.
US4765737A
Expired / Lapsed
US
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Cell size measurements using light in flow cytometry and cell sorting
Assignee: Cornell Research Foundation Inc
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Filing: 1987-03-30
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Granted: 1988-08-23
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Expires: 2007-03-30
Abstract
Abstract In a flow cytometer where the laser beam is larger than the size of each biological cell of a column of cells being passed through the laser beam at a high rate wherein new and improved method and means are used to calculate the size of the cells as sensed by low angle forward light scatter or secondary emission of fluorescent material on the surface of the cell by measuring the time-of-flight of the smaller cell across the laser beam, with the time-of-flight being measured by integrating an electrical signal from either the forward light scatter or the secondary flourescent emission from a single cell as it passes through the laser beam and deriving still another electrical signal measuring the peak amplitude of the first signal and then dividing the integrated signal by the peak signal and subtracting a constant electrical signal from that quotient.
Inventors
- 👤 William V. Harris
- 👤 Bruce R. Land
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 20:47
CPC Classifications
G; G01; G01N; G01N15/00; G01N15/10; G01N15/14; G01N15/1456; G01N15/1459; G01N15/01; G01N15/1404; G01N15/1409; G01N15/149; G01N15/02; G01N15/0205; G01N2015/0238; G01N2015/1497; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6402
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
21 words
A new and improved method of measuring the size of a biological cell in a flow cytometer, comprising the steps of:
Claim 2
Dependent Claim
21 words
A new and improved method of measuring the size of a biological cell in a flow cytometer, comprising the steps of:
Claim 3
Dependent Claim
228 words
Directing said biological cells in column flow at a constant velocity; 2. Passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell by a. deriving a first electrical signal from the low angle forward light scatter characteristic or secondary fluorescent emission from a single cell as it passes through the said laser beam; b. deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. deriving a third electrical signal having an amplitude equal to the peak (maximum) amplitude of said first electrical signal; d. deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each said biological cell plus the width of the laser beam; e. deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the said laser beam whereby the fifth electrical signal is directly proportional to the width of said biological cell.
Claim 4
Dependent Claim
228 words
Directing said biological cells in column flow at a constant velocity; 2. Passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell by a. deriving a first electrical signal from the low angle forward light scatter characteristic or secondary fluorescent emission from a single cell as it passes through the said laser beam; b. deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. deriving a third electrical signal having an amplitude equal to the peak (maximum) amplitude of said first electrical signal; d. deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each said biological cell plus the width of the laser beam; e. deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the said laser beam whereby the fifth electrical signal is directly proportional to the width of said biological cell.
Claim 5
Dependent Claim
16 words
A new and improved flow cytometer means for measuring the size of a biological cell, comprising:
Claim 6
Dependent Claim
16 words
A new and improved flow cytometer means for measuring the size of a biological cell, comprising:
Claim 7
Dependent Claim
244 words
Means for directing said biological cells in column flow at a constant velocity; 2. Means for passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Means for measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell or its nucleus, comprising: a. means for deriving a first electrical signal from the low angle forward light scatter characteristic or secondary fluorescent emission from each cell as it passes through the said laser beam; b. means for deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. means for deriving a third electrical signal having an amplitude equal to the peak amplitude of said first electrical signal; d. means for deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each said biological cell plus the width of the laser beam; e. means for deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the laser beam whereby the fifth electrical signal is directly proportional to the width of said biological cell.
Claim 8
Dependent Claim
244 words
Means for directing said biological cells in column flow at a constant velocity; 2. Means for passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Means for measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell or its nucleus, comprising: a. means for deriving a first electrical signal from the low angle forward light scatter characteristic or secondary fluorescent emission from each cell as it passes through the said laser beam; b. means for deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. means for deriving a third electrical signal having an amplitude equal to the peak amplitude of said first electrical signal; d. means for deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each said biological cell plus the width of the laser beam; e. means for deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the laser beam whereby the fifth electrical signal is directly proportional to the width of said biological cell.
Claim 9
Dependent Claim
43 words
The new and improved flow cytometer means of claim 2 wherein electrical sorting means are connected to the output of said means for deriving said fifth electrical signal to sort and collect each cell based on the magnitude of said fifth electrical signal.
Claim 10
Dependent Claim
28 words
The new and improved flow cytometer means of claim 3 wherein a computer is also connected to the output of said means for deriving said fifth electrical signal.
Claim 11
Dependent Claim
257 words
A new and improved flow cytometer means for measuring the size of a biological cell, comprising: 1. Means for directing said biological cells in column flow at a constant velocity; 2. Means for passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Means for measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell or its nucleus, comprising: a. means for deriving a first electrical signal from the secondary fluorescent emission from each cell as it passes through the said laser beam; b. means for deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. means for deriving a third electrical signal having an amplitude equal to the peak amplitude of said first electrical signal; d. means for deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each biological cell plus the width of the laser beam; e. means for deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the laser beam whereby the fifth electrical signal is directly proportional to the size of nucleus or cytoplasm of said biological cell.
Claim 12
Dependent Claim
257 words
A new and improved flow cytometer means for measuring the size of a biological cell, comprising: 1. Means for directing said biological cells in column flow at a constant velocity; 2. Means for passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Means for measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell or its nucleus, comprising: a. means for deriving a first electrical signal from the secondary fluorescent emission from each cell as it passes through the said laser beam; b. means for deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. means for deriving a third electrical signal having an amplitude equal to the peak amplitude of said first electrical signal; d. means for deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each biological cell plus the width of the laser beam; e. means for deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the laser beam whereby the fifth electrical signal is directly proportional to the size of nucleus or cytoplasm of said biological cell.
Claim 13
Dependent Claim
43 words
The new and improved flow cytometer means of claim 5 wherein electrical sorting means are connected to the output of said means for deriving said fifth electrical signal to sort and collect each cell based on the magnitude of said fifth electrical signal.
Claim 14
Dependent Claim
28 words
The new and improved flow cytometer means of claim 6 wherein a computer is also connected to the output of said means for deriving said fifth electrical signal.
Claim 15
Dependent Claim
254 words
A new and improved flow cytometer means for measuring the size of a biological cell, comprising: 1. Means for directing said biological cells in column flow at a constant velocity; 2. Means for passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Means for measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell, comprising: a. means for deriving a first electrical signal from the low angle forward light scatter characteristic from each cell as it passes through the said laser beam; b. means for deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. means for deriving a third electrical signal having an amplitude equal to the peak amplitude of said first electrical signal; d. means for deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each said biological cell plus the width of the laser beam; e. means for deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the laser beam whereby the fifth electrical signal is directly proportional to the width of said biological cell.
Claim 16
Dependent Claim
254 words
A new and improved flow cytometer means for measuring the size of a biological cell, comprising: 1. Means for directing said biological cells in column flow at a constant velocity; 2. Means for passing a laser beam having a cross section larger than each of said cells through said biological cells in column flow so that only one biological cell passes through said laser beam at a time; 3. Means for measuring the time of flight of each said biological cell through said laser beam deriving an electrical signal commensurate with the width and/or volume of each said biological cell, comprising: a. means for deriving a first electrical signal from the low angle forward light scatter characteristic from each cell as it passes through the said laser beam; b. means for deriving a second electrical signal having an amplitude determined by the time integral of said first electrical signal; c. means for deriving a third electrical signal having an amplitude equal to the peak amplitude of said first electrical signal; d. means for deriving a fourth electrical signal by dividing the said second electrical signal by the said third electrical signal therefore deriving an electrical signal commensurate with the width of each said biological cell plus the width of the laser beam; e. means for deriving a fifth electrical signal by subtracting from said fourth electrical signal a constant electrical signal proportional to the width of the laser beam whereby the fifth electrical signal is directly proportional to the width of said biological cell.
Claim 17
Dependent Claim
43 words
The new and improved flow cytometer means of claim 8 wherein electrical sorting means are connected to the output of said means for deriving said fifth electrical signal to sort and collect each cell based on the magnitude of said fifth electrical signal.
Claim 18
Dependent Claim
28 words
The new and improved flow cytometer means of claim 9 wherein a computer is also connected to the output of said means for deriving said fifth electrical signal.
References Cited (Backward Citations)
US4110043A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-08-23 | 1992-02-22 | 1992-08-23 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-08-23 | 1996-02-22 | 1996-08-23 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-08-23 | 2000-02-22 | 2000-08-23 | $7700.00 | Window Closed / Lapsed |