Digital sampling apparatus and methods for sorting particles

• US 8,198,092 B2
• Filed: 05/17/2011
• Issued: 06/12/2012
• Est. Priority Date: 03/28/2003
• Status: Expired due to Term

• Current Assignee: Inguran LLC
• Original Assignee: Inguran LLC
• Inventors: Durack, Gary, Wallace, Jeffrey D., Vandre, Gary P., Westfall, Lon A., Hatcher, Jeremy T., Nayak, Niraj V.
• Primary Examiner(s): Wallenhorst, Maureen
• Application Number:
  US13/109,640
• Publication Number:
  US 20110256575A1
• Time in Patent Office: 392 Days
• Field of Search: 422/67, 422/68.1, 422/73, 422/82.95, 422/82.08, 436/63, 436/164, 436/165, 436/43, 436/50, 436/55, 436/172, 435/2, 435/287.1, 435/286.1, 435/366, 435/288.7, 435/29
• US Class Current: 436/63
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 3/00 Condition responsive control processes apparatus therefor C12M1/36; controlling or regulating in general G05
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1406 Control of droplet point
• CPC Class Codes: G01N 2015/1415 Control of particle position
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/63 optically excited
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/48 Biological material, e.g. blood, urine G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence; Haemocytometers counting blood corpuscules distributed over a surface by scanning the surface G06M11/02
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: Y10T 436/10 : Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/115831 : Condition or time responsive
• CPC Class Codes: Y10T 436/12 : Condition responsive control
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2525 : Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A system and method for sorting a mixture of stained particles including a digital signal processor for analyzing and classifying the digital information generated from the particles and providing a sorting signal to a sorting system as a function of the analyzed and classified digital information.

Claims

1. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information include a series of waveform pulses, the waveform pulses being indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information wherein the digital signal processor includes instructions for detecting the waveform pulses corresponding to the digital information, instructions for extracting features in the detected waveform pulses, and instructions for discriminating the detected waveform pulses as a function of their extracted features.

2. A system as claimed in claim 1, wherein the digital signal processor detects portions of the digital information corresponding to the waveform pulses and classifies the detected portions, and wherein the digital signal processor provides the sorting signal as a function of said detected and classified portions.

3. A system as claimed in claim 1, wherein the particles are cells and characteristics A and B relate to physical characteristics of the cells.

4. A system as claimed in claim 3, wherein the stained particles are stained sperm cells, and characteristic A is indicative of a live X sperm cell and characteristic B is indicative of other than a live X cell (˜

X).

5. A system as claimed in claim 1, wherein the flow path is formed to contain particles following generally one after another in a series which includes sequential sets of particles, including first particle sets each including one or more particles having characteristic A, second particle sets each including one or more particles having characteristic B, and third particle sets each including two or more closely spaced particles at least one of which has characteristic A and at least one of which has characteristic B.

6. A system as claimed in claim 1, wherein the sorting system is a droplet sorting system or a photo-damage sorting system or a fluid-switching sorting system.

7. A system as claimed in claim 1, wherein the digital signal processor includes a pulse detection processor for detecting waveform pulses represented by the digital information, and in that the digital signal processor classifies the digital information as a function of the detected waveform pulses.

8. A system as claimed in claim 1, wherein the digital signal processor includes a sort processor responsive to the classifying for providing the sort signals to the sorting system.

9. A system as claimed in claim 8, wherein the sort processor enumerates the number of classified particles having characteristic A or having characteristic B.

10. A system as claimed claim 1, wherein the electromagnetic radiation source further comprises a pulsed illumination device.

11. A system as claimed claim 1, wherein the digital signal processor extracts features from the waveform pulses corresponding to the digital information and provides the sorting signal to the sorting system as a function of the extracted features.

12. The system as claimed in claim 11, wherein the digital signal processor includes a pulse detection processor for detecting waveform pulses represented by the digital information and for classifying the detected waveform pulses and the time varying output includes waveform pulses wherein the waveform pulses and the corresponding digital information are indicative of characteristic A or characteristic B and the a digital signal processor discriminates the digital information as indicative of characteristic A or as indicative of characteristic B and provides a sorting signal to the sorting system as a function of the discriminated digital information.

13. The system as claimed in claim 11, wherein the extracted features corresponds to one or more of the following features:

pulse area, pulse peak, pulse inner area, pulse width, pulse gaussianity, pulse lagging peak or pulse slope.

14. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired articles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information wherein the digital signal processor includes instructions for detecting pulses corresponding to the digital information, instructions for extracting features in the detected pulses, and instructions for defining a decision boundary discriminating between the extracted features representing characteristics A and the extracted features representing characteristic B.

15. The system as claimed in claim 14, wherein the digital signal processor adjusts the relative location of the decision boundary with respect to the extracted features representing characteristic A and with respect to the extracted features representing characteristic B as a function of at least one of the following:

a. the purity of the said at least one population with respect to either characteristic A particles or characteristic B particles; and

b. the quantity of characteristic A particles or characteristic B particles in the said at least one population relative to the total quantity of characteristic A particles or characteristic B particles in the flow path.

16. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired articles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor includes a data management processor for assembling the digital information into a continuous stream.

17. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired articles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor includes a pulse detection processor for detecting waveform pulses represented by the digital information, and in that the digital signal processor classifies the digital information as a function of a coefficient of variation of a population of the particles having characteristic A or as a function of a coefficient of variation of a population of the particles having characteristic B.

18. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B, wherein the analog to digital converter further comprises a continuous sampling rate;

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information; and

h. a filter for filtering the analog output at a frequency equal to or less than one half the continuous sampling rate of the analog to digital converter, wherein the analog to digital converter converts the filtered analog output into corresponding digital information, and in that the digital signal processor classifies the digital information as a function of a discrimination boundary.

19. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B, wherein the analog to digital converter further comprises a continuous sampling rate;

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information; and

h. a filter for filtering the analog output at a frequency equal to or less than one half the continuous sampling rate of the converter, and in that the converter converts the filtered analog output into corresponding digital information.

20. A system as claimed in claim 19, wherein the digital signal processor classifies the digital information as a function of a coefficient of variation of a population of the particles having characteristic A or as a function of a coefficient of variation of a population of the particles having characteristic B.

21. A system, for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B, wherein the analog to digital converter further comprises a continuous sampling rate and wherein the continuous sampling rate is about 105 MHz or higher; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information.

22. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor includes a feature extraction and discrimination processor for defining a decision boundary discriminating between extracted features representing characteristics A and extracted features representing characteristic B, and in that the feature extraction and discrimination processor extracts features represented by the digital information and classifies the features as a function of the decision boundary.

23. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor includes a feature extraction and discrimination processor for defining a decision boundary discriminating between extracted features representing characteristics A and extracted features representing characteristic B, and in that the feature extraction and discrimination processor extracts features represented by the digital information and classifies the features as a function of a coefficient of variation of a population of the particles having characteristic A or as a function of a coefficient of variation of a population of the particles having characteristic B.

24. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor classifies the digital information as a function of reducing a coefficient of variation of a population of the particles having characteristic A to be substantially equal to or less than a preset amount or as a function of minimizing a coefficient of variation of a population of the particles having characteristic B to be substantially equal to or less than a preset amount.

25. A system as claimed in claim 24, wherein the preset amount is either about 1.5% or less or is about 1.3% or less.

26. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor classifies the digital information such that a population of the particles having characteristic A and a population of the particles having characteristic B correspond to a computer model of three populations including a first model population of particles having characteristic A, a second model population of particles having characteristic B and a third model population of unaligned particles, said model estimating population statistics for each of the first, second and third model populations.

27. The system as claimed in claim 26, wherein the third model population includes two populations of unaligned particles, and the model estimates the population statistics for the two populations of unaligned particles.

28. A system, for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor determines background characteristics of the time-varying analog output from the digital information;

detects waveform pulses represented by the digital information as a function of the determined background characteristics; and

provides a sorting signal to the sorting system as a function of the detected waveform pulses.

29. A system as claimed in claim 28, wherein the digital signal processor employs an iterative procedure for determining a pulse detection threshold for defining the background characteristics, the iterative procedure including:

a. computing background statistic estimates from the digital information;

using the computed estimates to apply a pulse detection logic to the digital information in order to identify pulses indicative of characteristic A or characteristic B; and

b recomputing the estimates without using the digital information corresponding to the identified pulses; and

repeating the above procedure until the background statistic estimates converge or a fixed maximum number of iterations occurs.

30. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor generates initial discrimination parameters from the digital information, discriminates the digital information as a function of the initial discrimination parameters, and provides a sorting signal to the sorting system as a function of the discriminated digital information.

31. A system as claimed in claim 30, wherein the digital signal processor employs at least one of the following algorithms to generate the initial discrimination parameters:

k-Means, Fuzzy k-Means and Agglomerative Hierarchical.

32. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital processor employs a detection threshold for defining waveform pulses corresponding to the digital information, and wherein said detection threshold is a function of a background mean estimate and a standard deviation of the digital information computed within a moving window of samples ending with the current sample.

33. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information; and

h. a digital control which employs a statistical anomaly detection analysis, wherein digital information statistically anomalous from background characteristics of the digital information is considered part of a digital pulse.

34. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B, and wherein the electromagnetic radiation source further comprises a pulsed illumination device which is synchronized with the analog to digital converter sampling for synchronous sampling for illuminating the particles to produce corresponding waveform pulses; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information.

35. A system for sorting a mixture of stained particles, including stained particles having a characteristic A and stained particles having a characteristic B, the system comprising:

a. a fluid delivery system for delivering a fluid containing the stained particles in a flow path;

b. an electromagnetic radiation source for exciting fluorescence emissions from the stained particles having characteristic A and the stained particles having characteristic B in the flow path;

c. a photodetector for detecting the fluorescence emissions from the stained particles;

d. a processor in communication with the photodetector for classifying the stained particles according to their fluorescence emissions as either particles having characteristic A or particles having characteristic B;

e. a sorting system for sorting the stained particles according to the classification to provide at least one population containing desired particles;

f. an analog to digital converter for sampling a time-varying analog output from the photodetector and providing an output including digital information corresponding to the time-varying analog output wherein the time-varying analog output and the corresponding digital information are indicative of characteristic A or characteristic B; and

g. a digital signal processor for analyzing and classifying the digital information and providing a sorting signal to the sorting system as a function of the analyzed and classified digital information, wherein the digital signal processor extracts features from waveform pulses corresponding to the digital information and provides the sorting signal to the sorting system as a function of the extracted features, and wherein the extracted features include an approximation of a derivative of the pulse or slope of the pulse at a point of the pulse relative to an average peak height of the pulse.

36. The system as claimed in claim 35 wherein the point along the pulse corresponds to a point at which there is a difference between a first derivative of a pulse produced by particles having characteristic A and a first derivative of a pulse produced by particles having characteristic B.

37. The system as claimed in claim 35, wherein the time-varying analog output corresponds to a fluorescence emission pulse from the particles, and the point along the pulse either:

a. corresponds to a point at which a difference between a first derivative of a pulse produced by particles having characteristic A and a first derivative of a pulse produced by particles having characteristic B is at or near a maximum;

or

b. is a function of a peak height of the fluorescence emission pulses.

38. The system as claimed in claim 35, wherein the digital signal processor includes instructions for:

a. detecting waveform pulses represented by the digital information;

b. extracting features in the detected waveform pulses;

c. discriminating the detected waveform pulses as a function of their extracted features; and

d. defining a decision boundary discriminating between the extracted features representing characteristics A and the extracted features representing characteristic B.

39. The system as claimed in claim 35, wherein the digital signal processor further comprises instructions to extract features from the detected waveform pulses, and to discriminate the detected waveform pulses as a function of their extracted features.

40. A method for sorting a mixture of stained particles including stained particles having a characteristic A and stained particles having a characteristic B, the method including the steps of:

a. forming a stream containing stained particles;

b. generating an analog output with a photodetector detecting stained particles in the stream;

c. sampling the analog output from the photodetector with an analog to digital converter and generating corresponding digital information wherein the analog output is indicative of characteristic A or characteristic B;

d. analyzing and classifying the digital information, wherein the step of analyzing and classifying further comprises detecting waveform pulses represented by the digital information, extracting features of the waveform pulses from the digital information, and discriminating the detected waveform pulses as a function of their extracted features;

e. sorting the stained particles as a function of the analyzed and classified digital information; and

f. collecting the sorted particles to provide a population containing stained particles having a characteristic A and/or a population containing stained particles having a characteristic B.

41. A method as claimed in claim 40, wherein the particles are cells and characteristics A and B relate to physical characteristics of the cells.

42. A method as claimed in claim 41, wherein the particles are sperm cells, and characteristic A is indicative of a live X-chromosome bearing sperm cell and characteristic B is indicative of other than a live X-chromosome bearing cell (˜

X).

43. A method as claimed in claim 40 further comprising the step of forming the stream to contain particles following generally one after another in a series which includes sequential sets of particles, including first particle sets each including one or more particles having characteristic A, second particle sets each including one or more particles having characteristic B, and third particle sets each including two or more closely spaced particles at least one of which has characteristic A and at least one of which has characteristic B.

44. A method as claimed in claim 40, wherein the step of sorting uses a droplet sorting process, or a photo-damage sorting process, or a fluid-switching sorting process.

45. A method as claimed in claim 40 further comprising the step of defining a decision boundary discriminating between the extracted features representing characteristics A and the extracted features representing characteristic B.

46. A method as claimed in claim 40, wherein the step of sampling the analog output from the photodetector further comprises synchronously sampling the analog output from the photodetector.

47. A method as claimed in claim 40, wherein the step of classifying the digital information further comprises classifying the digital information as a function of minimizing a coefficient of variation of a population of the particles having characteristic A to be substantially equal to or less than a preset amount or as a function of minimizing a coefficient of variation of a population of the particles having characteristic B to be substantially equal to or less than a preset amount.

48. A method as claimed in claim 47, wherein the preset amount is about 1.5% or less.

49. A method as claimed in claim 40, wherein the step of classifying the digital information includes classifying the digital information such that a population of the particles having characteristic A and a population of the particles having characteristic B correspond to a computer model of three populations including a first model population of particles having characteristic A, a second model population of particles having characteristic B and a third model population of unaligned particles, said model estimating population statistics for each of the first, second and third model populations.

50. A method as claimed in claim 40, wherein the step of classifying the digital information further comprises classifying the digital information as a function of a coefficient of variation of a population of the particles having characteristic A or as a function of a coefficient of variation of a population of the particles having characteristic B.