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US5540494A
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Method and apparatus for determining absolute particle size, surface area and volume normalized fluorescence using forward angle light scatter intensity in flow cytometry
Assignee: Individual
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Filing: 1994-06-03
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Granted: 1996-07-30
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Expires: 2014-06-03
Abstract
Abstract A method and apparatus are described for flow cytometric absolute sizing of individual biological particles and for determining and reporting fluorescence data which is surface area or volume normalized.
Inventors
- 👤 Norman B. Purvis, Jr.
- 👤 Todd D. Giorgio
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 20:18
CPC Classifications
G; G01; G01N; G01N15/00; G01N15/10; G01N15/14; G01N15/02; G01N15/0205; G01N15/0211; G01N15/1012; G01N2015/1024
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
95 words
A method of determining surface and cytoplasmic characteristics of a biological particle using flow cytometry comprising the steps of: a. determining, directly from forward angle light scatter intensity information an absolute radius of said particle and measuring a fluorescence intensity of a fluorescent probe associated with said particle while said particle is in a conventional flow cytometer; b. calculating from said determined absolute particle radius an equivalent spherical surface area corresponding to said particle; and c. determining a surface area normalized fluorescence from a ratio of said fluorescence intensity to said equivalent spherical surface area.
Claim 2
Independent Claim
95 words
A method of determining surface and cytoplasmic characteristics of a biological particle using flow cytometry comprising the steps of: a. determining, directly from forward angle light scatter intensity information an absolute radius of said particle and measuring a fluorescence intensity of a fluorescent probe associated with said particle while said particle is in a conventional flow cytometer; b. calculating from said determined absolute particle radius an equivalent spherical surface area corresponding to said particle; and c. determining a surface area normalized fluorescence from a ratio of said fluorescence intensity to said equivalent spherical surface area.
Claim 3
Dependent Claim
45 words
The method of claim 1 further comprising the steps of calculating from said measured absolute particle radius an equivalent absolute spherical volume corresponding to said particle and determining a volume normalized fluorescence from a ratio of said fluorescence intensity to said equivalent absolute spherical volume.
Claim 4
Dependent Claim
55 words
The method of either of claim 1 or 2 where the step of measuring the absolute radius of said particle using forward angle light scatter intensity information from said flow cytometer includes the step of using Fraunhofer Diffraction Theory to derive an empirical equation relating the absolute particle radius to forward angle light scatter intensity.
Claim 5
Dependent Claim
89 words
The method of claim 3 wherein the empirical equation relating absolute particle radius to forward angle light scatter intensity comprises a fitted polynomial equation provided by determining, by a method of least squares, the best fit values of C, θ min , and θ max by comparing calculated forward angle light scatter intensity to actual measured forward angle light scatter intensity, where C is the flux per unit area of the incident light beam, and θ min and θ max are the minimum and maximum light scatter angles respectively.
Claim 6
Independent Claim
139 words
A method of determining an absolute radius of a biological particle using a conventional flow cytometer, said method comprising the steps of: a. characterizing particle sizing parameters of said flow cytometer by measuring in a light scatter detection system in said flow cytometer a total diffracted light scatter intensity associated with at least one calibration bead, said bead having a known size, shape, and refractive index; b. measuring in said light scatter detection system in said flow cytometer a total diffracted light scatter intensity associated with said particle; and c. correlating said diffracted light scatter intensity associated with said particle to an absolute particle radius by a particle radius correlation means, said correlation means optimized by empirically fitting, using Fraunhofer Diffraction Theory, a theoretical light scatter equation to measured total diffracted light scatter intensity associated with said calibration beads.
Claim 7
Independent Claim
139 words
A method of determining an absolute radius of a biological particle using a conventional flow cytometer, said method comprising the steps of: a. characterizing particle sizing parameters of said flow cytometer by measuring in a light scatter detection system in said flow cytometer a total diffracted light scatter intensity associated with at least one calibration bead, said bead having a known size, shape, and refractive index; b. measuring in said light scatter detection system in said flow cytometer a total diffracted light scatter intensity associated with said particle; and c. correlating said diffracted light scatter intensity associated with said particle to an absolute particle radius by a particle radius correlation means, said correlation means optimized by empirically fitting, using Fraunhofer Diffraction Theory, a theoretical light scatter equation to measured total diffracted light scatter intensity associated with said calibration beads.
Claim 8
Independent Claim
87 words
An apparatus for measuring biological particle size comprising: a. a flow cytometer having a forward angle light scatter intensity detection system; b. means for limiting minimum and maximum angles of collection of light scatter by said detection system; c. processor means for receiving from said detection system forward angle light scatter intensity data associated with a particle in said flow cytometer; d. said processor means further comprising correlation means to correlate an absolute radius directly from said forward angle light scatter intensity data associated with said particle.
Claim 9
Independent Claim
87 words
An apparatus for measuring biological particle size comprising: a. a flow cytometer having a forward angle light scatter intensity detection system; b. means for limiting minimum and maximum angles of collection of light scatter by said detection system; c. processor means for receiving from said detection system forward angle light scatter intensity data associated with a particle in said flow cytometer; d. said processor means further comprising correlation means to correlate an absolute radius directly from said forward angle light scatter intensity data associated with said particle.
Claim 10
Dependent Claim
35 words
The apparatus of claim 4, the correlation means comprising means to calculate an absolute particle radius associated with each of said particles in accordance with an equation empirically derived from a theoretical light scatter equation.
Claim 11
Dependent Claim
47 words
The apparatus of claim 4, the correlation means comprising data storage means for storing a lookup table which contains data mapping absolute particle radius to forward angle light scatter, said lookup table generated by empirically fitting a theoretical light scatter equation to actual measured absolute particle data.
Claim 12
Independent Claim
21 words
The apparatus of either of claims 7 or 8, wherein the theoretical light scatter equation is derived from Fraunhofer Diffraction Theory.
References Cited (Backward Citations)
US2816479A
Cited
US3695765A
Cited
US3710933A
Cited
US3873209A
Cited
US4263508A
Cited
US4360270A
Cited
US4857451A
Cited
US4794806A
Cited
US5041733A
Cited
US5138170A
Cited
US4765737A
Cited
US5007737A
Cited
US5090808A
Cited
US5056918A
Cited
US5104221A
Cited
US5105093A
Cited
US5325168A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1999-07-30 | 2000-01-29 | 2000-07-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2003-07-30 | 2004-01-29 | 2004-07-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2007-07-30 | 2008-01-29 | 2008-07-30 | $7700.00 | Window Closed / Lapsed |