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 - 231 . (canceled) 
     
     
         232 . A flow cytometry system for sorting sperm comprising:
 a) a nozzle for producing a stream comprising stained sperm cells in a sample and sheath fluid;   b) a pulsed laser for exciting stained sperm cells in the stream;   c) a detection system for detecting the emitted fluorescence of stained sperm cells;   d) a processing unit for differentiating X chromosome sperm from Y chromosome sperm;   e) an oscillator to which the stream is responsive for forming droplets;   f) a drop charge circuit to apply a charge to the droplets, wherein the charge applied to each droplet is based on the presence of an X-chromosome and/or Y-chromosome as determined by the processing unit;   g) a first and second deflection plate each disposed adjacent to a free fall area in which a droplet forms; and   h) at least one collector to collect sorted sperm.   
     
     
         233 . The flow cytometry system for sorting sperm as claimed in  claim 232 , further comprising a radiation beam manipulator selected from a group consisting mirrors, deflectors, beam splitters, prisms, refractive objects, lenses and filters. 
     
     
         234 . The flow cytometry system for sorting sperm as claimed in  claim 233 , wherein the detection system comprises a photomultiplier tube. 
     
     
         235 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein the stained sperm cells are stained with a fluorescent dye. 
     
     
         236 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein the fluorescent dye comprises Hoechst 33342. 
     
     
         237 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein the pulsed laser comprises an average power less than about 300 mW. 
     
     
         238 . The flow cytometry system for sorting sperm as claimed in  claim 232 , further comprising a beam splitter. 
     
     
         239 . The flow cytometry system for sorting sperm as claimed in  claim 232 , further comprising a high purity sorted population of said X chromosome bearing sperm and said Y chromosome bearing sperm. 
     
     
         240 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein said high purity is selected 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.   
     
     
         241 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein the pulsed laser comprises a pulse duration between about 5 to about 20 picoseconds. 
     
     
         242 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein the pulsed laser comprises a pulse resting period between about 0.5 to about 20 nanoseconds. 
     
     
         243 . The flow cytometry system for sorting sperm as claimed in  claim 232 , the pulsed laser has a repetition rate comprises between about 50 to about 200 MHz. 
     
     
         244 . The flow cytometry system for sorting sperm as claimed in  claim 243 , wherein the repetition rate comprises up to about 80 MHz. 
     
     
         245 . The flow cytometry system for sorting sperm as claimed in  claim 232 , wherein the sperm cells are stained with a reduced amount of stain. 
     
     
         246 . The flow cytometry system for sorting sperm as claimed in  claim 245 , wherein the reduced amount of stain comprises a percentage of stain selected from a group consisting of about 90%, about 80%, about 70% and about 60% of a maximum stain. 
     
     
         247 . The flow cytometry system for sorting sperm as claimed in  claim 232 , further comprising at least one shared resource, wherein said shared resource comprises a pulsed laser and a beam splitter.