Efficient haploid cell sorting flow cytometer systems

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.

1 - 59 . (canceled)  
     
     
         60 . A method of flow cytometry sample processing comprising the steps of: 
 establishing at least one sheath fluid;    flowing said at least one sheath fluid into at least two nozzles;    injecting at least one irradiatable sample into said at least one sheath fluid;    utilizing at least one shared resource to process said at least one irradiatable sample;    subjecting said irradiatable sample to radiation;    exciting said irradiatable sample with said radiation;    emitting fluorescence from said excited sample;    detecting an amount of said emitted fluorescence from each particle in said sample;    evaluating said amount of emitted fluorescence from each particle in said sample;    selecting an electrical condition to be associated with each particle of said sample in said sheath fluid flow;    charging a stream of said irradiatable sample and sheath fluid based upon deduced properties of each particle of said sample in said sheath fluid flow;    forming a charged drop;    isolating said charged drop from said sheath fluid flow;    deflecting said charged drops;    sorting said sample; and    collecting said sorted sample.    
     
     
         61 - 66 . (canceled)  
     
     
         67 . A method of flow cytometry sample processing according to  claim 60  wherein said step of subjecting said irradiatable sample to radiation comprises the step of subjecting said irradiatable sample to a continuous wave laser.  
     
     
         68 . A method of flow cytometry sample processing according to  claim 60  wherein said step subjecting said irradiatable sample to radiation comprises the steps of: 
 multiply subjecting said irradiatable sample to radiation for a first amount of time;    multiply terminating said radiation of said irradiatable sample for a second amount of time; and    multiply exciting said irradiatable sample with said radiation.    
     
     
         69 . A method of flow cytometry sample processing according to  claim 60  wherein said step of utilizing said at least one shared resource to process said at least one irradiatable sample comprises the step of utilizing one radiation source for subjecting said irradiatable sample in said at least two nozzles.  
     
     
         70 . (canceled)  
     
     
         71 . A method of flow cytometry sample processing according to  claim 60  and further comprising the step of splitting said radiation into at least two light beams.  
     
     
         72 . A method of flow cytometry sample processing according to  claim 71  wherein said step of splitting said radiation into at least two light beams comprises the step of subjecting said with a reduced power of radiation than which was originally emitted from a laser source.  
     
     
         73 . A method of flow cytometry sample processing according to  claim 72  wherein said step of subjecting said with a reduced power of radiation than which was originally emitted from a laser source comprises the step of selecting said reduced power from a group consisting of a half, a fourth, and an eighth of said originally emitted power.  
     
     
         74 . A method of flow cytometry sample processing according to  claim 60  wherein said step of detecting an amount of said emitted fluorescence from each particle in said sample comprises the step of quantitatively detecting an amount of said emitted fluorescence from each particle in said sample.  
     
     
         75 . (canceled)  
     
     
         76 . A method of flow cytometry sample processing according to  claim 74  wherein said step of injecting at least one irradiatable sample into said at least one sheath fluid comprises the step of injecting at least one irradiatable sperm cells into said at least one sheath fluid and wherein said step of quantitatively detecting an amount of said emitted fluorescence from each particle in said sample comprises distinguishing between a X chromosome bearing sperm and a Y chromosome bearing sperm wherein said X chromosome bearing sperm emits a different fluorescence from said Y chromosome.  
     
     
         77 . A method of flow cytometry sample processing according to  claim 60  wherein said step of sorting said sample comprises the step of rapidly sorting said sample.  
     
     
         78 . A method of flow cytometry sample processing according to  claim 77  wherein said step of rapidly sorting said sample cells comprises the step of sorting at a rate greater than 500 cells per second.  
     
     
         79 . A method of flow cytometry sample processing according to  claim 77  wherein said step of rapidly sorting said sample cells comprises the step of sorting at a rate selected from a group consisting of 
 greater than 1000 cells per second;    greater than 1500 cells per second;    greater than 2000 cells per second; and    greater than 3000 cells per second.    
     
     
         80 . A method of flow cytometry sample processing according to  claim 60  and further comprising the step of utilizing a beam manipulator.  
     
     
         81 . A method of flow cytometry sample processing according to  claim 80  wherein said step of utilizing a beam manipulator comprises the step of utilizing a beam manipulator selected from a group consisting of mirrors, deflectors, beam splitters, prisms, refractive objects, lenses and filters.  
     
     
         82 - 83 . (canceled)  
     
     
         84 . A method of flow cytometry sample processing according to  claim 60  wherein said step of injecting irradiatable sample comprises the step of staining said sample with fluorescent dye.  
     
     
         85 - 88 . (canceled)  
     
     
         89 . A method of flow cytometry sample processing according to  claim 84  wherein said step of staining said sample comprises the step of staining said sample for a reduced staining time.  
     
     
         90 . A method of flow cytometry sample processing according to  claim 89  wherein said step of staining for a reduced time comprises the step of staining said sample for less than about 40 minutes.  
     
     
         91 . A method of flow cytometry sample processing according to  claim 89  wherein said reduced staining time is selected from a group consisting of 
 less than about 35 minutes;    less than about 30 minutes;    less than about 25 minutes;    less than about 20 minutes;    less than about 15 minutes;    less than about 10 minutes; and    less than about 5 minutes.    
     
     
         92 . A method of flow cytometry sample processing according to  claim 60  wherein said step of exciting said irradiatable sample with said radiation comprises the step of sufficiently hitting said sample with said radiation to cause said irradiatable sample to emit fluorescence.  
     
     
         93 - 96 . (canceled)  
     
     
         97 . A method of flow cytometry sample processing according to  claim 60  wherein said step of collecting said sorted sample comprises the step of collecting at least two populations of sample particles.  
     
     
         98 . A method of flow cytometry sample processing according to  claim 97  wherein said step of collecting at least two populations of sample particles comprises the step of collecting a sorted population of X chromosome bearing sperm and collecting a sorted population of Y chromosome bearing sperm.  
     
     
         99 . A method of flow cytometry sample processing according to  claim 97  wherein said step of collecting said populations comprises the step of collecting said populations at a high purity.  
     
     
         100 . A method of flow cytometry sample processing according to  claim 99  wherein said step of collecting said populations at a high purity comprises the step of selecting said high purity from a group consisting of: 
 greater than 85% purity;    greater than 90% purity;    greater than 95% purity;    greater than 96% purity; and    greater than 98% purity.    
     
     
         101 . A method of flow cytometry sample processing according to  claim 99  wherein said step of collecting said populations at a high purity comprises the step of a providing a high resolution of said sorted sample.  
     
     
         102 . A method of flow cytometry sample processing according to  claim 101  wherein high resolution of said sorted sample is selected from a group consisting of: 
 greater than 7.0;    greater than 7.5;    greater than 8.0;    greater than 8.5;    greater than 9.0; and    greater than 9.2.    
     
     
         103 - 104 . (canceled)  
     
     
         105 . A method of flow cytometry sample processing according to  claim 97  wherein said step of collecting at least two populations of sample particles comprises the step of collecting said populations at a high collection rate.  
     
     
         106 . A method of flow cytometry sample processing according to  claim 105  wherein said high collection rate is selected from a group consisting of: 
 greater than 2400 particles per second;    greater than 2600 particles per second;    greater than 2900 particles per second;    greater than 3000 particles per second; and    greater than 3100 particles per second.    
     
     
         107 . A method of flow cytometry sample processing according to  claim 60  wherein said step of detecting an amount of said emitted fluorescence from each particle in said sample comprises the step of detecting at an event rate of between about 10,000 to about 60,000 particles per second.  
     
     
         108 . A method of flow cytometry sample processing according to  claim 60  wherein said step of subjecting said irradiatable sample to radiation comprises the step of initiating a sensing routine.  
     
     
         109 . A method of flow cytometry sample processing according to  claim 68  wherein said step of multiply subjecting said irradiatable sample to radiation for a first amount of time comprises the step multiply subjecting said irradiatable sample to radiation for a first amount of time between about 5 to about 20 picoseconds.  
     
     
         110 . A method of flow cytometry sample processing according to  claim 68  multiply terminating said radiation of said irradiatable sample for a second amount of time comprises the step of multiply terminating said radiation of said irradiatable sample for a second amount of time between about 0.5 to about 20 nanoseconds.  
     
     
         111 . A method of flow cytometry sample processing according to  claim 110  and further comprising providing a repetition rate between about 2 to about 10 microseconds.  
     
     
         112 . A method of flow cytometry sample processing according to  claim 111  wherein said step of providing a repetition rate comprises the step providing a repetition rate between 50-200 MHz.  
     
     
         113 . (canceled)  
     
     
         114 . A method of flow cytometry sample processing according to  claim 84  wherein said step of staining said samples with a fluorescent dye comprises the step of minimally staining said samples with a fluorescent dye.  
     
     
         115 . A method of flow cytometry sample processing according to  claim 114  wherein said step of minimally staining samples with a fluorescent dye comprises the step of allowing less stain to bind to said sample.  
     
     
         116 . A method of flow cytometry sample processing according to  claim 114  wherein said step of minimally staining said samples with a fluorescent dye comprises the step of providing a percentage of stain selected from a group consisting of about 90%, about 80%, about 70% and about 60% of a maximum stain.  
     
     
         117 . (canceled)  
     
     
         118 . A method of flow cytometry sample processing according to  claim 60  wherein said step of injecting at least one irradiatable sample into said at least one sheath fluid comprises injecting sperm cells selected from a group consisting of mammals, bovine sperm cells, equine sperm cells, porcine sperm cells, ovine sperm cells, camelid sperm cells, ruminant sperm cells, and canine sperm cells.  
     
     
         119 . A method of flow cytometry sample processing according to  claim 60  wherein said step of collecting said sorted sample comprises the step of collecting said sorted sample in a collector, wherein said collector is selected from the group consisting of multiple containers and a combined collector having a individual containers.  
     
     
         120 . A method of flow cytometry sample processing according to  claim 119  further comprising the step of providing a number of selected containers less than a number of nozzles.  
     
     
         121 . (canceled)  
     
     
         122 . A method of flow cytometry sample processing according to  claim 68  wherein said step of multiply subjecting said irradiatable sample to radiation for a first amount of time and said step of multiply terminating said radiation of said irradiatable sample for a second amount of time comprises the step of utilizing a pulsed laser.  
     
     
         123 . A method of flow cytometry sample processing according to  claim 122  wherein said step of utilizing a pulsed laser selected from a group consisting of Nd:YAG and Nd:YVO 4 .  
     
     
         124 . A method of flow cytometry sample processing according to  claim 60  and further comprising the step of individually controlling said at least two nozzles.  
     
     
         125 . A method of flow cytometry sample processing according to  claim 60  and further comprising the step of compositely controlling said at least two nozzles.  
     
     
         126 . A mammal produced through use of a sorted sperm cells produced with a flow cytometer system according to  claim 118 .  
     
     
         127 - 174 . (canceled)  
     
     
         175 . A flow cytometry system comprising: 
 at least one sheath fluid port to introduce a sheath fluid;    at least one sample injection element having an injection point through which an irradiatable sample may be introduced into said sheath fluid;    at least two nozzles located in part below said at least one injection point;    an oscillator to which said sheath fluid is responsive;    a radiation emitter;    a particle sample fluorescence detector;    a processing unit connected to said particle sample fluorescence detector;    a drop charge circuit to apply an electrical condition to a stream of said irradiatable sample cells and sheath fluid;    a first and second deflection plate each disposed on opposite sides of a free fall area in which a drop forms, wherein said first and second deflection places are oppositely charged; and    a particle sample collector.    
     
     
         176 - 231 . (canceled)