Sperm processing methods
View Patent ↗A method of analyzing particles in a fluid stream including focusing a generally elliptical beam spot to a width less than the size of the particles. As particles pass through the beam spot electromagnetic radiation from the particles which is then detected and converted into electrical signals indicative of characteristics of the particles. The electrical signals may be digitally sampled to provide digital information which may be analyzed to extract information and to classify the particles.
1. A method of analyzing chromosomal DNA characteristics of animal sperm cells in a fluid stream having a direction of flow, each sperm cell having a head with a nucleus comprising a localized chromosome region containing at least one chromosome, said nucleus having a length in the direction of stream flow, said method comprising:
focusing a pulsed beam of electromagnetic radiation on the fluid stream as a generally elliptical spot having a length along a major axis extending generally at right angles to the direction of stream flow and a width along a minor axis extending generally parallel to the direction of stream flow, the width of the beam spot being less than the length of said nucleus, said sperm cells, when passing through said spot, emit electromagnetic radiation, including emissions from said chromosome regions indicative of chromosomal DNA characteristics of the regions, said pulsed beam of electromagnetic radiation being emitted at a frequency to impinge on the sperm cells multiple times as they pass through the beam spot;
detecting and converting at least some of said emissions from the chromosome regions into time-varying analog electrical signals indicative of said chromosomal DNA characteristics;
digitally sampling the time-varying analog signal and providing an output including digital information corresponding to said time-varying analog signal;
analyzing the digital information to extract information indicative of a characteristic of the time-varying analog signals; and
classifying at least some of the sperm cells as having a particular chromosomal DNA characteristic based at least in part on said extracted information.
2. The method of claim 1 , wherein said chromosomal DNA characteristics are indicative of the sex of the sperm cells.
3. The method of claim 2 , wherein said sperm cells are bovine sperm cells.
4. The method of claim 3 , wherein said chromosome region is localized within an area of the nucleus extending no more than about 20% of the nucleus length on either side of a longitudinal center of the nucleus.
5. The method of claim 3 , wherein said chromosome region is localized within an area of the nucleus extending no more than about 10%-15% of the nucleus length on either side of a longitudinal center of the nucleus.
6. The method of claim 1 , wherein the digitally sampling step comprises synchronously sampling the time-varying analog signal.
7. The method of claim 1 , wherein said spot width is less than about 3.0 μm.
8. The method of claim 7 , wherein said spot length is greater than about 100 μm.
9. The method of claim 1 , further comprising exerting a force on the sperm cells in the stream tending to bring them into a desired orientation before the stream passes through said beam.
10. The method of claim 1 , further comprising droplet sorting said sperm cells according to their classification based on said chromosomal DNA characteristics.
11. The method of claim 1 , further comprising sorting said sperm cells according to their classification based on said chromosomal DNA characteristics with a photo-damaging laser.
12. The method of claim 1 , further comprising fluid-switching sorting sperm cells according to their classification based on said chromosomal DNA characteristics.
13. The method of claim 1 , wherein said characteristic of the time-varying analog signal comprises a slope of the time-varying analog signal at a point of the signal compared against a threshold value.
14. A method of analyzing particles in a fluid stream having a direction of flow, said method comprising:
focusing a pulsed beam of electromagnetic radiation on the fluid stream as a generally elliptical spot having a length along a major axis extending generally at right angles to the direction of stream flow and a width along a minor axis extending generally parallel to the direction of stream flow, the width of the beam spot being less than the size of said particles, said particles, when passing through said spot emit electromagnetic radiation, said pulsed beam of electromagnetic radiation being emitted at a frequency to impinge on the particles multiple times as they pass through the beam spot;
detecting and converting at least some of said emissions into time-varying analog electrical signals indicative of characteristics of the particles;
digitally sampling the time-varying analog signal and providing an output including digital information corresponding to said time-varying analog signal;
analyzing the digital information to extract information indicative of a characteristic of the time-varying analog signals; and
classifying at least some of the particles as having a particular characteristic based at least in part on said extracted information.
15. The method of claim 14 , wherein the digitally sampling step comprises synchronously sampling the time-varying analog signal.
16. The method of claim 14 , further comprising droplet sorting said particles according to their classification based on said extracted information.
17. The method of claim 14 , further comprising sorting said particles according to their classification based on said extracted information with a photo-damaging laser.
18. The method of claim 14 , further comprising fluid-switching sorting particles according to their classification based on said extracted information.
19. The method of claim 14 , wherein the characteristic of the time-varying analog signal comprises a slope of the time-varying analog signal at a point of the signal compared against a threshold value.
20. The method of claim 14 , wherein the characteristic of the time-varying analog signal comprises an area of a waveform pulse representing the time-varying analog signal.
21. The method of claim 20 , wherein the characteristic of the time-varying analog signal comprises an area of the waveform pulse representing the time-varying analog signal comprising 60% to 20% of the area of the waveform pulse centered on a peak of the waveform pulse.
22. The method of claim 20 , wherein the characteristic of the time-varying analog signal comprises an area of the waveform pulse representing the time-varying analog signal comprising 30% to 20% of the area of the waveform pulse centered on a peak of the waveform pulse.