Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
DC CAFC
First Claim
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1. A method of flow cytometry sperm processing comprising the steps of:
- establishing a sheath fluid;
flowing said sheath fluid into at least one nozzle;
providing a percentage of a fluorescent dye stain as compared to a standard control, said percentage of stain selected from a group consisting of about 90%, about 80%, about 70%, and about 60% of maximum stain;
staining sperm cells with said percentage of said fluorescent dye stain to provide irradiatable stained sperm cells;
injecting said irradiatable stained sperm cells into said sheath fluid;
utilizing one radiation source;
splitting a radiation into at least two beams;
directing each of said beams to said at least one nozzle and said sperm cells;
multiply subjecting said irradiatable sperm cells to said radiation for a first amount of time;
multiply terminating said radiation of said irradiatable sperm cells for a second amount of time;
multiply exciting said irradiatable sperm cells with said radiation;
wherein said step of multiply subjecting said irradiatable sperm cells to said radiation for said first amount of time and said step of multiply terminating said radiation of said irradiatable sperm cells for said second amount of time and said step of multiply exciting said irradiatable sperm cells with said radiation are performed in a sequential manner then repeated in order at a repetition rate;
emitting fluorescence from said excited sperm cells;
detecting an amount of said emitted fluorescence from each of said sperm cells;
evaluating said amount of emitted fluorescence from each of said sperm cells;
selecting an electrical condition based on the amount of emitted and detected fluorescence from each of said sperm cells to be associated with each of said sperm cells in said sheath fluid flow;
charging a stream of said irradiatable sperms cell and sheath fluid based on the amount of emitted and detected fluorescence from each of said sperm cells in said sheath fluid flow;
forming a charged drop having one of said sperm cells located therein;
isolating said charged drop from said sheath fluid flow;
deflecting said charged drops;
sorting said sperm cells; and
collecting said sorted sperm cells.
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Abstract
A flow cytometry system (1) for sorting haploid cells, specifically irradiatable sperm cells, with an intermittingly punctuated radiation emitter (56). Embodiments include a beam manipulator (21) and even split radiation beams directed to multiple nozzles (5). Differentiation of sperm characteristics with increased resolution may efficiently allow differentiated sperm cells to be separated higher speeds and even into subpopulations having higher purity.
624 Citations
69 Claims
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1. A method of flow cytometry sperm processing comprising the steps of:
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establishing a sheath fluid; flowing said sheath fluid into at least one nozzle; providing a percentage of a fluorescent dye stain as compared to a standard control, said percentage of stain selected from a group consisting of about 90%, about 80%, about 70%, and about 60% of maximum stain; staining sperm cells with said percentage of said fluorescent dye stain to provide irradiatable stained sperm cells; injecting said irradiatable stained sperm cells into said sheath fluid; utilizing one radiation source; splitting a radiation into at least two beams; directing each of said beams to said at least one nozzle and said sperm cells; multiply subjecting said irradiatable sperm cells to said radiation for a first amount of time; multiply terminating said radiation of said irradiatable sperm cells for a second amount of time; multiply exciting said irradiatable sperm cells with said radiation; wherein said step of multiply subjecting said irradiatable sperm cells to said radiation for said first amount of time and said step of multiply terminating said radiation of said irradiatable sperm cells for said second amount of time and said step of multiply exciting said irradiatable sperm cells with said radiation are performed in a sequential manner then repeated in order at a repetition rate; emitting fluorescence from said excited sperm cells; detecting an amount of said emitted fluorescence from each of said sperm cells; evaluating said amount of emitted fluorescence from each of said sperm cells; selecting an electrical condition based on the amount of emitted and detected fluorescence from each of said sperm cells to be associated with each of said sperm cells in said sheath fluid flow; charging a stream of said irradiatable sperms cell and sheath fluid based on the amount of emitted and detected fluorescence from each of said sperm cells in said sheath fluid flow; forming a charged drop having one of said sperm cells located therein; isolating said charged drop from said sheath fluid flow; deflecting said charged drops; sorting said sperm cells; and collecting said sorted sperm cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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42. A flow cytometry system for sperm comprising:
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a sheath fluid port to introduce a sheath fluid; a sample injection element having an injection point through which irradiatable sperm cells may be introduced into said sheath fluid, said irradiatable sperm cells comprises stained sperm cells having a percentage of stain as compared to a standard control, said percentage of stain selected from a group consisting of about 90%, about 80%, about 70%, and about 60% of maximum stain; a nozzle located in part below said injection point; an oscillator to which said a sheath fluid is responsive; a pulsed laser; a beam splitter; a sperm cell fluorescence detector to detect fluorescence from said irradiatable sperm cells located in a stream flowing out of said nozzle;
said sperm cell fluorescence detector located near said stream of irradiatable sperm cells;a processing unit connected to said sperm cell fluorescence detector to evaluate detected fluorescence from said irradiatable sperm cells; a drop charge circuit located near said stream of irradiatable sperm cells flowing out of said nozzle to apply an electrical condition to a stream of said irradiatable sperm cells and sheath fluid, said drop charge circuit is responsive to said processing unit and applies said electrical condition based on said evaluated detected fluorescence; a first and second deflection plate responsive to said processing unit, each plate disposed on opposite sides of a free fall area in which a drop forms, wherein said first and second deflection plates are oppositely charged; and a sperm cell collector located below said first and second deflection plates. - View Dependent Claims (43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69)
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Specification
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Litigation Data
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Current AssigneeXY, LLC (Inguran LLC)
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Original AssigneeXY Incorporated (Inguran LLC)
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InventorsGilligan, Thomas B., Evans, Kenneth M.
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Primary Examiner(s)WALLENHORST, MAUREEN
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Application NumberUS11/442,735Publication NumberTime in Patent Office1,461 DaysField of Search436/63, 436/164, 436/165, 436/43, 436/50, 436/55, 436/172, 422/67, 422/68.1, 422/73, 422/82.05, 422/82.08, 435/2, 435/4, 435/287.1, 702/21US Class Current436/63CPC Class CodesC12N 5/0612 sorting of gametes, e.g. ac...C12Q 3/00 Condition responsive contro...G01N 15/1433 using image recognitionG01N 15/147 the analysis being performe...G01N 15/149 specially adapted for sorti...G01N 33/5005 involving human or animal c...Y10T 436/12 Condition responsive control
