Efficient haploid cell sorting flow cytometer systems

Abstract

A flow cytometer system for sorting haploid cells, specifically sperm cells, may include an intermittingly punctuated radiation emitter, such as a pulsed laser. Embodiments include a beam manipulator and even split radiation beams directed to multiple nozzles. Differentiation of sperm characteristics with increased resolution may efficiently allow differentiated sperm cells to be separated at higher speeds and even into subpopulations having higher purity.

1 - 231 . (canceled) 
     
     
         232 . A method of sorting sperm cells comprising the steps of:
 producing a stream of stained sperm cells and sheath fluid;   exciting the stained sperm cells within the stream with a pulsed laser;   detecting an emitted fluorescence from each of the stained sperm cells;   distinguishing between X chromosome bearing sperm and Y chromosome bearing sperm based on the emitted fluorescence in response to the pulsed laser;   isolating sperm cells within droplets;   charging the droplets based upon the emitted fluorescence of the sperm cells contained within each droplet;   deflecting the charged droplets; and   collecting the deflected droplets to form at least one sorted sperm population.   
     
     
         233 . The method of  claim 232  wherein the step of producing a stream of stained sperm cells and sheath fluid further comprises the step of injecting stained sperm cells in a sheath fluid within a nozzle. 
     
     
         234 . The method of  claim 232  wherein said step of detecting an amount of emitted fluorescence from each of the stained sperm cells comprises the step of detecting an amount of emitted fluorescence from each of the sperm cells with a detection system utilizing a photomultipler tube. 
     
     
         235 . The method of  claim 232  wherein the step of exciting stained sperm cells within the stream with a pulsed laser comprises subjecting the stained sperm cells to a laser pulse for a pulse duration between about 5 to about 20 picoseconds. 
     
     
         236 . The method of  claim 232  wherein the step of exciting stained sperm cells within the stream with a pulsed laser comprises providing a pulse resting period between about 0.5 to about 20 nanoseconds. 
     
     
         237 . The method of  claim 232  wherein the step of exciting stained sperm cells within the stream with a pulsed laser further comprises providing a repetition rate between 50-200 MHz. 
     
     
         238 . The method of  claim 232  wherein the step of exciting stained sperm cells within the stream with a pulsed laser comprises providing an average power radiation of less than 300 mW. 
     
     
         239 . The method of  claim 232  further comprising the step of staining the sperm cells with a reduced amount of fluorescent dye. 
     
     
         240 . The method of  claim 232  wherein the step of staining the sperm cells with a reduced amount of fluorescent dye comprises providing a percentage of stain selected from a group consisting of about 90%, about 80%, about 70% and about 60% of a maximum stain. 
     
     
         241 . The method of  claim 232  wherein the sperm cells are selected from a group consisting of non-human mammals, bovine sperm cells, equine sperm cells, porcine sperm cells, ovine sperm cells, camelid sperm cells, ruminant sperm cells, and canine sperm cells. 
     
     
         242 . The method of  claim 232  further comprising the step of utilizing at least one shared resource to sort the sperm cells. 
     
     
         243 . The method of  claim 242 , wherein the step of utilizing at least one shared resource to sort the sperm cells comprises the steps of:
 utilizing one pulsed laser;   splitting the pulsed laser into at least two beams; and   directing each of the beams to a different stream of stained sperm cells and sheath fluid.   
     
     
         244 . The method of  claim 232  further comprising the step of utilizing said collected sorted sperm for insemination of female non-human mammals. 
     
     
         245 . A non-human mammal produced through use of a sorted sperm cells produced with a flow cytometer system according to  claim 232 . 
     
     
         246 . A flow cytometry system for sorting sperm comprising:
 a. a nozzle for producing a stream of stained sperm cells 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 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.   
     
     
         247 . The flow cytometry system for sorting sperm as claimed in  claim 246  further comprising a radiation beam manipulator selected from a group consisting mirrors, deflectors, beam splitters, prisms, refractive objects, lenses and filters. 
     
     
         248 . The flow cytometry system for sorting sperm as claimed in  claim 247  wherein the detection system comprises a photomultipler tube. 
     
     
         249 . The flow cytometry system for sorting sperm as claimed in  claim 246  wherein the stained sperm cells are stained with a fluorescent dye. 
     
     
         250 . The flow cytometry system for sorting sperm as claimed in  claim 246  wherein the fluorescent dye comprises Hoechst 33342. 
     
     
         251 . The flow cytometry system for sorting sperm as claimed in  claim 246  wherein the pulsed laser comprises an average power less than about 300 mW. 
     
     
         252 . The flow cytometry system for sorting sperm as claimed in  claim 246  further comprising a beam splitter. 
     
     
         253 . The flow cytometry system for sorting sperm as claimed in  claim 246  further comprising a high purity sorted population of said X chromosome bearing sperm and said Y chromosome bearing sperm. 
     
     
         254 . The flow cytometry system for sorting sperm as claimed in  claim 246  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. 
 
     
     
         255 . The flow cytometry system for sorting sperm as claimed in  claim 246  wherein the pulsed laser comprises a pulse duration between about 5 to about 20 picoseconds. 
     
     
         256 . The flow cytometry system for sorting sperm as claimed in  claim 246  wherein the pulsed laser comprises a pulse resting period between about 0.5 to about 20 nanoseconds. 
     
     
         257 . The flow cytometry system for sorting sperm as claimed in  claim 246  the pulsed laser has a repetition rate comprises between about 50 to about 200 MHz. 
     
     
         258 . The flow cytometry system for sorting sperm as claimed in  claim 257  wherein the repetition rate comprises up to about 80 MHz. 
     
     
         259 . The flow cytometry system for sorting sperm as claimed in  claim 246  wherein the sperm cells are stained with a reduced amount of stain. 
     
     
         260 . The flow cytometry system for sorting sperm as claimed in  claim 259  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. 
     
     
         261 . The flow cytometry system for sorting sperm as claimed in  claim 246  further comprising at least one shared resource, wherein said shared resource comprises a pulsed laser and a beam splitter.