U.S. patent application number 10/556981 was filed with the patent office on 2006-11-23 for efficient haploid cell sorting flow cytometer systems.
Invention is credited to Todd A. Cox, Kenneth M. Evans, Thomas B. Gilligan, Tae Kwang Suh.
Application Number | 20060263829 10/556981 |
Document ID | / |
Family ID | 33476853 |
Filed Date | 2006-11-23 |
United States Patent
Application |
20060263829 |
Kind Code |
A1 |
Evans; Kenneth M. ; et
al. |
November 23, 2006 |
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.
Inventors: |
Evans; Kenneth M.; (College
Station, TX) ; Gilligan; Thomas B.; (Fort Collins,
CO) ; Suh; Tae Kwang; (Fort Collins, CO) ;
Cox; Todd A.; (Fort Collins, CO) |
Correspondence
Address: |
SANTANGELO LAW OFFICES, P.C.
125 SOUTH HOWES, THIRD FLOOR
FORT COLLINS
CO
80521
US
|
Family ID: |
33476853 |
Appl. No.: |
10/556981 |
Filed: |
May 15, 2004 |
PCT Filed: |
May 15, 2004 |
PCT NO: |
PCT/US04/15457 |
371 Date: |
November 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60471509 |
May 15, 2003 |
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|
Current U.S.
Class: |
435/7.2 ;
800/24 |
Current CPC
Class: |
G01N 2015/149 20130101;
G01N 15/147 20130101; G01N 33/5005 20130101; G01N 15/1475 20130101;
C12Q 3/00 20130101; Y10T 436/12 20150115; C12N 5/0612 20130101 |
Class at
Publication: |
435/007.2 ;
800/024 |
International
Class: |
G01N 33/567 20060101
G01N033/567; A01K 67/027 20060101 A01K067/027 |
Claims
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.sub.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)
Description
I. FIELD OF THE INVENTION
[0001] The present invention relates to a flow system for particle
analysis. More specifically, the invention relates to the use of a
pulsed laser on a flow system for particle analysis which results
in more accurate quantification of measurable properties of
individual particles. It may be of particular interest in analyzing
populations of very similar particles, at high speeds, allowing
more efficient separation of particles into two or more different
populations. The invention is particularly useful in the
application of separating live X-chromosome bearing and
Y-chromosome bearing sperm of all mammals at higher speeds, better
purities and with equal or better sperm health outcomes, meaning
less damage to sperm. The invention may contribute significant
improvements to the economics of sperm sorting.
II. BACKGROUND OF THE INVENTION
[0002] Lasers can be used to deliver light to biological or
non-biological particles and emission spectra can be used for the
analysis of particle characteristics. In some instances, this can
be applied such as where a particle is self florescent or self
color absorbing, is associated by affinity, avidity, covalent
bonds, or otherwise to another molecule which may be colored or
fluorescent, may be associated to another molecule which is colored
or fluorescent through a specific biological or modeled
macromolecular interaction, such as an antibody binding event or a
nucleic acid oligomer or poynucleic acid hybridization event, may
obtain color or fluorescence such as through an enzymatic synthesis
event, an enzymatic attachment or cleavage reaction, enzymatic
conversion of a substrate, association of a florescent molecule
with a nearby quencher, the reaction of a product in certain local
proton (pH) or NADH or NADPH or ATP or free hydride (H--) or bound
hydride R--(H--) concentrations, or may gain color or fluorescense
by way of a variety of methods to associate emitted or absorbed
light (electromagnetic radiation EMR).
[0003] Conventional lasers can generate a strong, perhaps intense,
source of light. Through coherence properties of the beam such
light may travel very long distances, perhaps across reflective
mirrors which may change the angle of the light illumination beam,
perhaps through prisms or refractive objects or lenses which may
split it into two or more beams of equal or differing intensity, or
may defocus, perhaps expand, or focus, perhaps concentrate, the
beam. Such light may also be affected by filters which may reduce
the net energy of the beam. Most lasers also allow the modulation
of light intensity, perhaps watts, in the beam by adjustment of an
input current from a power supply to the light generating
element.
[0004] In some applications, conventional lasers used in the
analysis and quantification of biological objects can be combined
with sensitive light detectors that may be as simple, such as a
photographic film or paper, or may be more complex, such as a
photomultiplier tube. Often, a light detector may collect only
information about a cumulative amount of light, perhaps
electromagnetic radiation, EMR, or it may collect and report on the
dynamic changes in intensity of light or EMR hitting all of, or
portions of, localized regions of, or positions on the detector
surface. The light detector may also involve use of a photoelectric
coupling device, which may allow the energy of photons absorbed on
the EMR by the light detector to be converted to current
proportional to the incident light or EMR on the light detector
surface. The photoelectric coupling device can even be integrated
into an electronic circuit with an amplifier which may increase the
signal or create gain such that the fluctuations or perhaps
summation of amplified current may be available to an analog or
digital logic circuit. Designs may also transmit a signal or data
set to a user of a particle analysis instrument and this signal may
be proportional to the static, cumulative, or perhaps even dynamic
intensity of the light or EMR incident upon the detector.
[0005] In certain uses of laser light to analyze biological
particles, a detector may measure the change in intensity of the
source light after incidence upon a particle(s) being analyzed
using a reference beam which takes a path without incidence upon
the particle(s). In other uses of laser light, modified or
unmodified particles take up a fraction of the illumination light
or EMR and may emit light of a different frequency. In many cases,
the presence of emission light or EMR of a certain wavelength cam
be used to identify or to quantify characteristics associated with
specific particles, or quantitatively measure the amount or number
of the specific biological particles present in the sample or in a
specific region of or position in the sample.
[0006] In some cases, it can be useful to accurately determine very
small differences in the illumination light or emission light from
two very similar biological particles (for example an X-chromosome
bearing sperm cell versus a Y-chromosome bearing sperm cell). These
small differences can be analyzed by way of serial presentation of
perhaps 50,000 separate emission events per second in a liquid
stream. These can also be thousands of separate emissions from
molecules (nucleic acids or proteins as examples) on an array field
allowing analysis of genetic, genomic, proteomic, or glycomic
libraries.
[0007] The traditional type of laser used for the analysis of
particles in flow cytometry is a continuous wave (CW) laser. Often
this provides a beam of constant intensity. However, in some
instances, CW lasers can have particular disadvantages for
applications as discussed here. The beam can result in modification
or destruction of the sample being observed. For example, with
respect to sperm cells, irradiation can result in lower fertility
of the sperm cells. Second, in some instances when the laser beam
continuously operates, it may be desirable to have a method of
interrupting the beam if it is moved from a first location of
incidence to a second location of incidence without illumination of
intermediate areas.
[0008] In U.S. Pat. No. 5,596,401 to Kusuzawa, a pulsed laser may
be used for imaging an object, such as a cell, in a flow cytometer.
This disclosure may be related to improvements in the capture of
images from particles such as coherence lowering modulations.
Kusuzawa may teach a use of a continuous wave laser for particle
detection and imaging.
[0009] In U.S. Pat. No. 5,895,922 and U.S. Patent Application No.
2003/0098421 to Ho, pulsed laser light may be used to illuminate
and detect hazardous biological particles dispersed in an airflow
stream. The invention may include an ultraviolet laser light and
looking for the emission of fluorescence from potentially hazardous
biological particles. This disclosure may teach the disadvantages
of a laser diode apparatus.
[0010] U.S. Pat. No. 6,177,277 to Soini, describes employing a
two-photon excitation and/or confocal optimal set-up. The invention
may relate to the use of confocal optics to reduce an analysis
volume to about 10% of standard analysis volume in a flow
cytometer. A pulsed laser may provide short pulses of intense light
and may allow the simultaneous absorption of two photons so that a
wavelength of illuminating light beam may be longer than an emitted
single photon bursts. Background signal may be reduced by use of a
filter. The invention may include dual signal processing. The
invention as described in Soini, may be beneficial in the analysis
of small particles such as erythrocytes and bacterial cells.
[0011] In U.S. Pat. No. 6,671,044 to Ortyn, a special analysis
optics and equipment may be used in an imaging flow cytometer. The
Ortyn disclosure may include analyzing a sex of fetal cells in
maternal blood as a method for determining the sex of a child
during early pregnancy. Ortyn may indicate that analysis rates from
an imaging flow cytometer may be restricted to theoretically
maximizing at 500 cells per second.
[0012] With respect to particle analysis using laser light, the
present invention discloses technology which addresses each of the
above-mentioned problems.
[0013] For the purposes of this invention, a rapidly pulsed, high
intensity pulsed laser may be used. This laser may deliver short
pulses of high intensity perhaps lasting about 5-20 picoseconds,
followed by intervals between pulses which are 100-1000 times as
long as the pulses or about 0.5-20 nanoseconds. The light may have
very high peak intensities over the period of about 5-20
picoseconds, and low net energies over the period of about 2-10
microseconds.
[0014] Flow cytometry, using a high-speed cell analysis, or
high-speed cell analysis and sorting instrument, often relies on a
laser light source to illuminate a stream of fluid in which
particles are entrained. Particles may be caused to flow by a point
of illumination at a rapid rate, often in the range of 500 to
100,000 particles per second. Often the light from the illuminating
laser source is of constant intensity. The particles in the
analysis stream may be of the same size, and may spend the same
amount of time within the area of illumination. The amount of light
illuminating each particle in a large population of particles
analyzed in series may be identical. A detector may be capable of
measuring scattered light, or other types of light emitted by the
particle as a result of auto-fluorescence or fluorescence
associated with a chemical dye, dye complex, or conjugated dye
which may be targeted to one or more types of molecular species
contained on or within particles in the population and can
determine the identity of a particle and, in some cases, make a
measurement of the quantity of a specific molecular target
associated with the particle. A specific molecular structure on or
even within a particle may be characterized and a quantitative
measurement of the amount of associated molecular structure on or
even within a particle, may yield information which may be used as
a basis for sorting out or separating one type of particle from
another.
[0015] In a flow cytometer, there may be a very short time duration
between the exact moment that a particle is illuminated and the
exact moment that a physical manipulation or an electrical
condition, may be triggered to elicit separation of a specific
particle from a stream containing various particles. An example of
a physical manipulation may be charging of a droplet. A specific
duration may be called a drop delay period, and the duration may be
perhaps as brief as about 100 microseconds or perhaps as long as
about 10 milliseconds, and may even be about 1 millisecond. In the
case of particle sorting, information may be detected from each
particle, computational analysis of the information may be
determined, and comparison of the computation to a gating value or
perhaps even a selection criteria may be accurately performed
within a time period shorter than a duration of the drop delay.
[0016] Flow cytometer systems may be useful for measuring an
average amount of a specific molecule present on or even within a
population of particles. Past systems may not have measured the
exact amount of a specific molecule on or even within a population
of particles. Factors which can contribute to inaccurate
measurements of single particles may include the saturation of a
stain or even a conjugate to a particle, variation in the quanta of
illumination light, effects from the shape of a particle, and
perhaps even electronic noise in the detection apparatus.
[0017] An example of a particularly challenging problem is the
sorting of X-chromosome bearing and Y-chromosome bearing sperm of
mammals at high processing rates and high sorting purities. The
population of sperm in most mammals is about 50% X-chromosome
bearing and about 50% Y-chromosome bearing. A stain, such as
Hoechst 33342, may form complexes with double stranded DNA. A
measurement of total Hoechst 33342-DNA complex in each sperm may
correlate to the total amount of DNA in each sperm. In general,
mammals have larger X chromosomes than Y chromosomes and may have a
differential between total DNA contents of X-chromosome bearing
over Y-chromosome bearing sperm for various mammals. Such
differentials may include: human having about 2.8%; rabbit having
about 3.0%; pig having about 3.6%; horse having about 3.7%; cow
having about 3.8%; dog having about 3.9%; dolphin having about
4.0%; and sheep having about 4.2%. The differentials may correlate
to a relative difference of intensities emitted from a stained
sperm being sorted for the purpose of separation of X-chromosome
bearing and Y-chromosome bearing sperm.
[0018] Significant achievements have been made in developing
staining conditions to stain DNA in live sperm with Hoechst 33342,
such as, the use of dual orthogonal detection systems to determine
sperm orientation, the use of hydrodynamic fluidics to increase the
numbers of correctly oriented sperm, the setting of gain on
detectors, and even the use of high-speed electronics. In the most
efficient use of said achievements, it may be possible in most
mammals to simultaneously sort sperm into two populations,
X-chromosome bearing, and Y-chromosome bearing, at rates of 2500
per second or higher. It may also be possible to sort sperm to
purities of 90% or even higher. There may be, however, a distinct
problem in that at rates faster than 2500 per second, the purity of
the sample may decrease.
[0019] This problem may be understood due to the observation that
the co-efficient of variation (CV) in possibly even the best sperm
sorting procedures may be between about 0.7%-1.5%, and with poor
conditions can even be between about 2%-5%. Since the difference in
DNA between X-chromosome bearing sperm and Y-chromosome bearing
sperm in mammals may be as low as 2.8% as seen in humans and as
high as 7.5% as can be seen in chinchillas, the CV may be lower
than the DNA differential in order to achieve a large enough
separation of the two populations. Humans have one of the lowest
known DNA differentials and may have some of the lowest known
maximum purities in sorting. It may be desirable to improve
procedures which can reduce the CV.
[0020] A method which has been shown to improve the CV may be to
use higher intensities of laser light illumination. For example, it
is known to use continuous wave lasers to sort various sperm
species with between about 100-200 milliwatts of laser
illumination, and possibly with about 150 milliwatts. It has been
observed that doubling or tripling the intensity and increasing the
power to about 300-500 mW can improve the CV. An improved CV can be
most apparent by analysis of the "split" between two peaks on a
histogram. Yet, there may be problems associated with an increase
of intensity or perhaps even an increase of power with a continuous
wave laser. In the case of analyzing a Hoechst 33342 DNA complex
with a continuous wave laser, the light source may be near a UV
spectrum and may have some ionizing effect upon the DNA complex.
Ionizing may then cause changes to the DNA. Accordingly, sperm
sorted with high intensities continuous wave lasers such as 300-500
mW may not be as fertile. Another problem may include the energy
that it may take to power a continuous laser to deliver about 150
mW of energy at near UV spectrum. Continuous wave lasers may
require 10,000 mW or perhaps even more of power. Since there may
already be a large amount of electrical power required to run a
continuous wave laser at 150 mW, a much larger amount of power may
be required to run a continuous wave laser at higher powers.
Furthermore, a tube life of ion lasers may be reduced when
operating at higher powers. An additional problem with the use of
continuous wave lasers may be that the CV may drop significantly
when using lower powers such as between about 20-80 mW.
[0021] In embodiments, the present invention provides flow
cytometer designs which may incorporate the use of 2 or more flow
nozzles, and even as many as dozens of flow nozzles, possibly
operated by a single sorting instrument. Fields such as
microfluidics, optics, electronics, and even parallel processing
may be explored. In other embodiments the present invention
includes the use of beam splitters to create multiple light beams.
Yet, a major problem facing the development of reliable flow
analysis and flow sorting in parallel may be the high intensity of
laser light needed for analysis at each nozzle. This problem is
particularly relevant for applications which require a very low CV
in measurement of identical particles.
[0022] There is a need to provide flow systems for the analysis and
sorting of particles that require a low CV value, yet may require
higher laser light intensities, yet higher intensities may have
negative effects on sperm and require higher power. In the search
for solutions to the problems in flow systems for the analysis and
sorting of particles, the field of pulsed lasers represents a
possible solution.
[0023] Surprisingly, even though sperm sorted on a high speed flow
cytometer may be damaged by UV light between about 300-500 mW, it
is now shown in this invention that powers between about 100-500 mW
may not be damaging to sperm if they are delivered in pulses. In
embodiments, this may include a pulse having a peak intensity
possibly as much as 1000 times higher than the intensity of a
continuous wave laser. Pulsed lasers may be designed as
quasi-continuous wave lasers and may have fast repetition rates
such as between about 50-200 Megahertz and even up to 80 Megahertz.
In embodiments, pulses may be between about 5-20 picoseconds.
Pulsed lasers may be ideal for providing pulsed light to a stream
of particles being analyzed in a flow cell or a flow cytometer.
Particles analyzed in flow cytometers with event rates possibly
between 10,000-100,000 Hertz, and even between 20,000-60,000 Hertz,
may be illuminated from a few hundred pulses from a laser having
repetition rates near 80,000 Hertz. Each pulse may provide an
intense amount of energy.
[0024] There may be certain industrial uses of flow cytometers, as
preparative instruments, which may be economically limited by the
traditional methods of processing. It may be desirable to provide
systems which facilitate parallel processing for industries such as
those that rapidly process mammalian ejaculates for the production
of large numbers of live sperm for insemination, those that process
blood samples for the recovery of specified cells such as fetal
cells, white blood cells, stem cells, hematopoetic cells from bone
marrow, and the like. In an embodiment of the present invention,
special forms of pulsed laser light can allow a single laser to
illuminate a plurality of nozzles, perhaps even while not reducing
the CV of the samples analyzed.
[0025] As a result, by the use of special forms of pulsed laser
light, further improvements in the speed and sample purity can be
seen. These types of lasers may be essential in the design and
development of new flow cytometers perhaps having multiple sorting
streams as well.
III. SUMMARY OF INVENTION
[0026] Accordingly, a broad object of the invention may provide a
particle analysis system having a pulsed laser which can be
operated at a low power.
[0027] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which may allow
detection of smaller differences in illumination or emission to
differentiate a particle characteristic.
[0028] Yet another broad object of the invention can be to provide
a particle analysis system having a pulsed laser which allows
differentiated particles to be separated into subpopulations having
a higher incidence of the desired characteristic.
[0029] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which allows
multiple particle differentiation systems to be run simultaneously
using a single laser.
[0030] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which affords
greater resolving power than conventional particle analysis systems
using a CW laser.
[0031] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which generates from
fluorochromes upon irradiation greater light intensity than
conventional particle analysis systems using a CW laser.
[0032] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which allows
differentiated particles to be separated into subpopulations at a
greater rate than conventional particle analysis systems using a CW
laser.
[0033] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which allows sperm
cells of any species of mammal to be differentiated with increased
resolution into X-chromosome or Y-chromosome bearing
subpopulations. The benefits of this object of the invention may
allow differentiation of sperm cells having: less DNA bound
fluorochrome, less residence time in staining protocols, greater
elapsed storage time prior to sorting, or perhaps even less
affinity to stain due to having been frozen prior to staining
protocols.
[0034] Another broad object of the invention can be to provide a
particle analysis system having a pulsed laser which allows sperm
cells to be separated into X-chromosome or Y-chromosome bearing
subpopulations having higher purity or separated into X-chromosome
or Y-chromosome bearing subpopulations at a greater number per
second.
[0035] Yet another broad object of the invention can be to provide
a miniaturized and parallel flow cytometer which allows a multiple
of nozzles sorting in tandem to be positioned on the same
apparatus, that may allow increases in the production rate of
sorting, by increasing the number of nozzles which are sorting on a
single apparatus.
[0036] Naturally, further independent objects of the invention are
disclosed throughout other areas of the specification.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 provides a block diagram of an embodiment of a
particle analysis system invention which includes beam manipulators
such as optical elements, splitters, filters, directional or the
like.
[0038] FIG. 2 provides other embodiments of beam manipulators.
[0039] FIG. 3 represents a fluidically connected system according
to certain embodiments of the invention.
[0040] FIG. 4 is a simplified representation of a representative
pulse of radiation that may used in some embodiments.
[0041] FIG. 5 provides illustrations of characteristics of a pulsed
illumination beam.
[0042] FIG. 6 provides illustrations which may differentiate
characteristics of a pulsed radiation beam from a conventional
continuous wave radiation beam.
[0043] FIG. 7 shows an embodiment of the invention representing a
sorting process having multiple nozzles.
[0044] FIG. 8 is a drawing of a flow sort embodiment of the
invention.
[0045] FIG. 9 provides an expanded diagram showing various
embodiments of a multiple nozzle assembly.
[0046] FIG. 10 depicts embodiments of certain time intervals and
light energy quantities which may be derived from particular
properties of pulsed light.
[0047] FIG. 11 is a comparison of a pulsed laser radiation beam and
a continuous wave laser radiation beam.
[0048] FIG. 12 is a representation of an embodiment for a sensing
routine.
[0049] FIG. 13 is a representation of a comparison of aggregate
data from various trial data.
[0050] FIG. 14 provides histograms and bivariant plots of
X-chromosome bearing and Y-chromosome bearing subpopulations of
sperm cells using a flow sort embodiment of the invention which
provides a 20 mW pulsed laser beam incident to the sperm cells
analyzed.
[0051] FIG. 15 provides histograms and bivariant plots of
X-chromosome bearing and Y-chromosome bearing subpopulations of
sperm cells using a flow sort embodiment of the invention which
provides a 60 mW pulsed laser beam incident to the sperm cells
analyzed.
[0052] FIG. 16 provides histograms and bivariant plots of
X-chromosome bearing and Y-chromosome bearing subpopulations of
sperm cells using a flow sort embodiment of the invention which
provides a 90 mW pulsed laser beam incident to the sperm cells
analyzed.
[0053] FIG. 17 provides histograms and bivariant plots of
X-chromosome bearing and Y-chromosome bearing subpopulations of
sperm cells using a flow sort embodiment of the invention which
provides a 130 mW pulsed laser beam incident to the sperm cells
analyzed.
[0054] FIG. 18 provides histograms and bivariant plots of
X-chromosome bearing and Y-chromosome bearing subpopulations of
sperm cells using a flow sort embodiment of the invention which
provides a 160 mW pulsed laser beam incident to the sperm cells
analyzed
[0055] FIG. 19 provides histograms of sperm cells analyzed with a
flow sort embodiment of the invention compared to a histogram of
sperm cells from the same sample analyzed with conventional CW flow
sort technology.
V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0056] As mentioned earlier, the present invention includes a
variety of aspects, which may be combined in different ways. The
following descriptions are provided to list elements and describe
some of the embodiments of the present invention. These elements
are listed with initial embodiments, however it should be
understood that they may be combined in any manner and in any
number to create additional embodiments. The variously described
examples and preferred embodiments should not be construed to limit
the present invention to only the explicitly described systems,
techniques, and applications. Further, this description should
further be understood to support and encompass descriptions and
claims of all the various embodiments, systems, techniques,
methods, devices, and applications with any number of the disclosed
elements, with each element alone, and also with any and all
various permutations and combinations of all elements in this or
any subsequent application.
[0057] Referring primarily to FIG. 1, the present invention
provides, in embodiments, a radiation emitter used with particle
analysis systems. In some embodiments of the invention, a radiation
emitter (12) or even an intermittingly punctuated radiation element
may convert electrical current into photons of radiation of a
specific wavelength and may generate radiation or even laser light
for analysis of non-biological or biological particles. Radiation
(11) may enter a chamber or region (22) in which it may be
modulated or even modified, such as but not limited to establishing
a coherent wave form. Radiation may be modified by a beam
manipulator (21) such as optical elements before it may illuminate
a particle or even a particle sample(s).
[0058] In some embodiments, beam manipulators (21) such as optical
elements may be used and may be located along a light path. Beam
manipulators may include mirrors, optically reflective or even
refractive mirrors, partially mirrored surfaces, deflectors, beam
splitters, refractive objects, lenses, filters, prisms, lenses, or
the like. Beam manipulators may modify or even modulate a pulsed
laser light by focusing, condensing, de-focusing, expanding,
splitting, or the like. Radiation may be split into multiple beams
of equal or perhaps even unequal intensity. A radiation beam
manipulator may influence a radiation beam such as by adapting a
beam or changing a beam as a particular situation may be
needed.
[0059] With respect to some embodiments of the invention,
positional control elements (24) can provide positional control
over a mirror, lens, prism, filter, or other optically relevant
components. A positional control element may influence the angle of
reflection or even refraction of a beam, and may even ultimately
direct a final position of a pulsed laser light beam on a particle
or particle sample(s). A positional control element may be a
mechanical device which may move a mirror along a path, or it may
even be a ratcheting or even a stepped device which can provide
large numbers of predefined angles for an optical element. A
positional control element(s) (24) may be used at any point in a
pulsed laser and even a pulsed light assembly or apparatus to
modify a beam of pulsed laser light. An oscillator may provide a
constant vibration in an optical element and may define a frequency
and amplitude.
[0060] In order to provide splitting of radiation into at least two
light beams, the present invention may include a beam splitter.
This may subject sperm cells to a reduced power of radiation than a
power of radiation with was originally emitted from a laser source
such as radiation emitter. Examples of reduced radiation may
include splitting a radiation beam in half, in a fourth of said
originally emitted power or even in an eighth of the originally
emitted power, as may be represented by FIG. 2. Of course, there
are many options in which to reduce power and all are intended to
be included within the scope of this disclosure. With a pulsed
laser, for example producing a 1000 mW output split into 8 equal
beams of approximately 120 mW, it may be possible to have 8 flow
cytometry nozzles sorting together from one light source.
[0061] As to other embodiments of the invention, independent beams
of pulsed laser light may be equal or may even be unequal in
intensity. Split beams can be derived from a source beam of pulsed
laser light by the action of optical splitters. As shown by FIG. 2,
a beam splitter (31) such as a prism can divide a beam of radiation
into at least two beams of the same frequency and pulse rate, but
may have equal or even unequal intensities. Split beams of light
may have intensities which can be each less intense than, or even
additively close to an equivalent to the original beam which
entered a splitter or prism. A complex beam splitter or prism,
which with multifaceted, three dimensional geometries can split a
beam of pulsed laser light into more than one, certainly two,
possibly dozens or even hundreds of independent beams of pulsed
laser light.
[0062] Complex beam splitters (34) may include a three dimensional
packed array of simple splitters or even prisms which can create
combined three dimensional geometries of refraction and reflection
and can even split a beam of radiation into more than one,
certainly two, possibly dozens or even hundreds of independent
beams of pulsed laser light. This may differ from a complex beam
splitter in that an array of simple splitters and even prisms may
allow individual geometries of each component, refractive index of
each component, which may allow a much larger number of options. A
rotational beam splitter (35) can be rotated on an axis,
potentially at very high speeds, such that an extremely large
number of pulsed laser light beams may be created to the point
where one can no longer speak of actual beams, but rather of each
pulse moving in an independent direction, slightly different that
the prior pulse. Beam splitters or optical splitters may be used at
any point along beam of pulsed laser light.
[0063] Another example of a beam manipulator (21) may include
filters can be placed in a beam path to modulate or modify a
property of a pulsed laser light, and may even reduce the net
energy of a particular beam. A large number of filters may be used
in series or even in parallel across many different beams of pulsed
laser light.
[0064] A radiation emitter (12) or even a intermittingly punctuated
radiation emitter may be embedded in an integrated fashion into a
particle analysis or particle separation system. Alternately, with
respect to some embodiments of the invention, a pulsed laser source
may be independent and splitters, as described above, may be used
to split an original beam of light to provide illumination light to
numerous independently operating particle analysis components.
[0065] It is understood that a fundamental unit of illumination may
be one single pulse of a laser light, and that each pulse or laser
light may be split numerous times. Through splitting, filtration or
even both, a net energy of any given pulse illuminating a
biological object or sample may be perhaps as small as a single
photon of light or perhaps as large as an original pulse energy
emitted from a laser unit.
[0066] It is also understood that the use of splitters, which can
divide laser light beams into two or more beams, may increase the
number of light beams that can illuminate particles, such as
biological objects. In embodiments, a number of pulsed light beams
per second that can be directed toward particles can be multiplied
with a splitter. It is also understood that the pulses per second
may be altered to a desired number of pulses per second, timing of
the pulse, and even position the pulse. A pulsed light may be
distributed by an apparatus to possibly millions of particles or
particle sample(s) which may be located in different positions.
Through the use of harmonically synchronized oscillations,
rotations, and even geometries, many pulses per second may be
delivered to the same particle, sample, or even biological objects.
It is particularly understood that, in embodiments, systems may be
established with recurring illumination events.
[0067] An embodiment of the present invention may include a system
fluidicly connected system, such as a flow cytometer system, as
seen in FIG. 3. This may be representative of a multiple number of
flow cytometry systems that are linked as one system.
[0068] Radiation emitters and even intermittingly punctuated
radiation emitters, as described in more detail below, may provide
one, two, three or perhaps even more radiation beams having
specific frequencies, wavelengths, intensities, and even watts to
illuminate the type of particles to be analyzed. An intermittingly
punctuated radiation emitter may multiply subject radiation to, for
example, sperm cells for a first amount of time and may multiply
terminate radiation of sperm cells for a second amount of time
(41). This may be represented by FIG. 4.
[0069] In embodiments, a first amount of time (40) may include an
amount of time radiation occurs and this time may be between about
5 to about 20 picoseconds. A second amount of time (41) may be a
radiation off time and may be between about 0.5 to about 20
nanoseconds. Of course, other amounts of time for each of a first
amount and a second amount of time may be used and all are
understood to be included within the scope of this invention. A
cycle of a first and second time may be understood as a repetition
(42). Each repetition may include a time of about 2 to about 10
microseconds, yet the repetition may vary. In embodiments, a
repetition rate may include between about 50 to about 200 MHz and
may even include a rate up to about 80 MHz. Other repetition rates
are possible and all are meant to be included within the scope of
this disclosure. In embodiments, a radiation emitter may be a
Nd:YAG, Nd:YVO.sub.4, or the like radiation emitters.
[0070] FIG. 4 shows parameters taken into consideration when
discussing the differences between continuous wave (CW) lasers or
even gas and pulsed lasers or even solid state lasers. As shown by
FIG. 4, an intermittingly punctuated radiation emitter (56) may
emit radiation that may not be continuous, yet be in short regular
pulses which may have a duration time or a first amount of time
(40). Following a pulse, there may be a dark period or a second
amount of time (41) in which no light may be generated. The total
elapsed time between two pulses, a repetition rate, may therefore
be the duration time in addition to the dark period. A pulse line
width and dark period may be similar in length, and it could be
postulated that a laser may actually be illuminating a sample for
somewhat less than "half of the time". Alternatively, a pulse line
width may be much smaller than a dark period, and thus a sample may
be illuminated for only a very small fraction of the time.
[0071] Peak energy (36) and a full amount of energy or joules
delivered from one pulse of laser light is represented in FIG. 5.
Fractional amounts (37) of that energy can be split as described
above or put through a neutral density filter. Importantly, one may
diminish the amount of light in one pulse used to illuminate a
particle by dividing or filtering the beam. For example, a 350 mW
beam can be split into 10 equal beams of approximately 35 mW to run
10 independent analysis machines from a single source laser. In
practice, the quality of analysis at 35 mW must afford information
regarding the characteristics of the particle illuminated, and for
commercial applications perhaps afford at least the same amount of
information as when particles illuminated with a CW UV laser
running at the standard of 150 mW. FIG. 5 may further help in an
understanding of the block diagram represented in FIG. 4. Each
radiation pulse may be reduced in energy as previously
discussed.
[0072] An energy density or even watts may be needed to achieve
maximum light emission (38) from a particle upon illumination as
shown by FIG. 6. It has been contemplated that light input of a
continuous wave laser, however, may be so low that a particle may
never be fully saturated with illumination (43). An emission light
(44) from particles illuminated by a CW laser at a given energy
intensity may be constant, as the source light may be constantly
refreshing the particle to a certain partial saturation value. By
comparison, emission light (39) from the same particle which has
been illuminated with pulses may be greater than from a CW laser.
Pulses may be short and may have illumination light intensity many
orders of magnitude higher than the illumination level of a CW
laser. It has also been speculated that a fate of light emission
from a particle or particle sample(s) during the dark period may be
dependent upon the half-life of emission for the illuminated
particle and may even be dependent upon the length of time of the
dark period. In the case where a half life may be as long or longer
than the dark period, the emission could remain close to maximum
during the entire time across all pulses delivered to the
sample.
[0073] It should also be pointed out that if instead of reducing
the input energy by splitting or filtering, one instead uses
movement of mirrors and reflectors, one may reduce the number of
pulses delivered to a biological sample to as low as one pulse,
while retaining the very strong intensity of the pulse. Thus, it is
a unique aspect of the invention to provide movement of the full
strength pulsed laser beam across a plurality of particles which
may for example be entrained in a fluid stream which passes through
a flow cytometry nozzle or located on an array or matrix (such as a
DNA or protein microarray), or a combination of both, as would be
the case of a single laser being oscillated so that it illuminates
a small number of flow cytometry nozzles in close proximity.
[0074] Now referring primarily to FIG. 8, irradiatable sperm cells
may be introduced through a sperm sample injection element (4)
which may act to supply irradiatable sperm cells for flow cytometry
analysis. Irradiatable sperm cells may be deposited within a nozzle
(5) in a manner such that the particles or cells are introduced
into a sheath fluid (3). A nozzle may be located in part below an
injection point of sperm cells. A sheath fluid (3) may be supplied
by a sheath fluid source (46) through a sheath fluid port (2) so
that irradiatable sperm cells and sheath fluid may be concurrently
fed through a nozzle (5). Accordingly, the present invention may
provide establishing a sheath fluid and flowing a sheath fluid into
a nozzle, and injecting irradiatable sperm cells into a sheath
fluid as shown in FIG. 8.
[0075] Further, in embodiments, the present invention may include
subjecting irradiatable sperm cells radiation. Radiation may be
produced from a radiation emitter (12) as discussed previously. In
embodiments the radiation emitter may be a intermittingly
punctuated radiation emitter or may even be a continuous wave
laser.
[0076] In embodiments, the present invention may include multiply
subjecting sperm cells to radiation having a wavelength appropriate
to activate fluorescence in a sperm cell. The invention may include
a fluorescence activation wavelength. Such wavelength may include
about 355 nm. Of course, this may include any wavelength that may
be needed to activate fluorescence. Such other wavelengths may
include 350 mm, 360 nm and other wavelengths and all are meant to
be included within the scope of this disclosure.
[0077] In embodiments, the present invention may include
sufficiently hitting a sperm with radiation to cause an
irradiatable sperm to emit fluorescence. This may include providing
radiation at certain wavelength, power, energy and the like that is
enough to cause an irradiatable sperm to emit fluorescence.
[0078] In embodiments, the present invention may provide for
exciting irradiatable sperm cells that have been subjected to
radiation. When in an excited state, the cells may emit
fluorescence. In embodiments, irradiatable sperm cells may be
multiply excited with radiation. This may include radiation that is
emitted from a intermittingly punctuated radiation emitter.
[0079] A pulse of laser light may illuminate particles or even a
particle sample(s) at a specific location with an EMR frequency or
Hertz, timing such as a clock, intensity or even watts, and even
net energy or joules. The particles may absorb the pulsed light,
may get excited and may even emit light of the same frequency as
that of the pulsed laser light, such as a scatter or may even emit
a light of a difference frequency or even fluorescence. The exact
nature of the amount of energy absorbed by a particle may be
related to the chemical properties of the particle, the chemical
properties of any objects attached to or closely associated with
the particles, the physical chemical properties of the particle
environment, such as biological segregations including membranes,
organelles, solutes, pH, temperature, osmolality, colloidal
character, or the like, and may even be related to the frequency
and intensity of the laser light illuminating the particle. An EMR
light emission from a particle, characterized by a wavelength and
quantity, can provide highly accurate information about the status
of a particle when a pulse of illuminating light is incident.
Depending on the nature of the particle and the particle
environment, the particle may then emit a florescent light signal,
and may do so over a certain period of time defined by a half-life
of emission. Typically, a number of pulses of laser light can
illuminate a particle or particle sample in a specified period of
time, and there can be a corresponding dynamically changing
emission of light over the same period, or a time period after
illumination.
[0080] Emitted fluorescence from each of the sperm cells may be
detected with a detection system (23). A detection system may
include a fluorescence detector (7) which may be connected to a
processing unit (15). While processing the emitted fluorescence,
the present invention may include evaluating the emitted signals.
Evaluation of emitted fluorescence may include how much
fluorescence may be emitted possibly by comparison between the
cells or may even possibly be compared to a predetermined number.
The present invention may include, in embodiments, selecting an
electrical condition to be associated with each of the sperm cells
in a sheath fluid flow. An electrical condition may be a charge,
voltage or any electrical condition. A drop charge circuit (8) may
charge a stream of cells and sheath fluid based upon deduced
properties of each of the excited cells. For example, this may be
to charge all of the X-chromosome bearing sperm cells with a
positive charge, and charging all of the Y-chromosome bearing sperm
cells with a negative charge. Of course, while the disclosure
focuses primarily upon sperm cells, other particles may be analyzed
as discussed in the various embodiments disclosed.
[0081] A charged drop may be formed and isolated in a free fall
area. A drop may be based upon whether a desired cell does or does
not exist within that drop. In this manner the detection system may
act to permit a first and second deflection plates (18) to deflect
drops based on whether or not they contain the appropriate cell or
other item. The deflection plates may be disposed on opposite sides
of a free fall area in which a drop may form and the deflection
plates may be oppositely charged. As a result, a flow cytometer may
act to sort cells by causing them to land in one or more
collectors. Accordingly, by sensing some property of the cells or
other items, a flow cytometer can discriminate between cells based
on a particular characteristic and place them in the appropriate
collector. In some embodiments, X-bearing sperm droplets are
charged positively and deflected in one direction, and Y-bearing
sperm droplets are charged negatively and deflected the other way.
A wasted stream which may be unsortable cells may be uncharged and
may be collected in collector, an undeflected stream into a suction
tube or the like.
[0082] In this manner, a sheath fluid may form a sheath fluid
environment for the sperm cells to be analyzed. Since the various
fluids are provided to the flow cytometer at some pressure, they
may flow out of nozzle (5) and exit through a nozzle orifice (47).
By providing some type of oscillator (6) which may be very
precisely controlled through an oscillator control (45), pressure
waves may be established within the nozzle and transmitted to the
fluids exiting the nozzle at nozzle orifice. Since an oscillator
may act upon the sheath fluid, a stream (19) exiting the nozzle
orifice (47) can eventually and regularly forms drops (9). Because
the particles or cells are surrounded by the fluid stream or sheath
fluid environment, the drops (9) may entrain within them
individually isolated particles or cells, such as sperm cells with
respect to some embodiments of the invention.
[0083] In other embodiments, since the drops can entrain particles
or cells, the flow cytometer can be used to separate particles,
cells, sperm cells or the like based upon particle or cell
characteristics. This is accomplished through a particle or cell
detection system (23). The particle or cell detection system
involves at least some type of detector (7) which responds to the
particles or cells contained within fluid stream. The particle or
cell sensing system may cause an action depending upon the relative
presence or relative absence of a characteristic, such as
fluorochrome bound to the particle or cell or the DNA within the
cell that may be excited by an irradiation source such as a
radiation emitter (12) generating an irradiation beam to which the
particle can be responsive. While each type of particle, cell, or
the nuclear DNA of sperm cells may be stained with at least one
type of fluorochrome different amounts of fluorochrome bind to each
individual particle or cell based on the number of binding sites
available to the particular type of fluorochrome used. With respect
to spermatozoa, the availability of binding sites for Hoechst 33342
stain is dependant upon the amount of DNA contained within each
spermatozoa. Because X-chromosome bearing spermatozoa contain more
DNA than Y-chromosome bearing spermatozoa, the X-chromosome bearing
spermatozoa can bind a greater amount of fluorochrome than
Y-chromosome bearing spermatozoa. Thus, by measuring the
fluorescence emitted by the bound fluorochrome upon excitation, it
is possible to differentiate between X-bearing spermatozoa and
Y-bearing spermatozoa.
[0084] As a result, the flow cytometer acts to separate the
particle or cells by causing them to be directed to one or more
collection containers. For example, when the analyzer
differentiates sperm cells based upon a sperm cell characteristic,
the droplets entraining X-chromosome bearing spermatozoa can be
charged positively and thus deflect in one direction, while the
droplets entraining Y-chromosome bearing spermatozoa can be charged
negatively and thus deflect the other way, and the wasted stream
(that is droplets that do not entrain a particle or cell or entrain
undesired or unsortable cells) can be left uncharged and thus is
collected in an undeflected stream into a suction tube or the like
as discussed in U.S. Pat. No. 6,149,867 to Seidel, hereby
incorporated by reference herein. Naturally, numerous deflection
trajectories can be established and collected simultaneously.
[0085] Irradiatable sample cells may include a sample cell that is
capable of emitting rays of light upon illumination. This may or
may not include having stain molecules attached to a sample
particle. Some particles may have features that allow them to emit
fluorescence naturally without having to add a stain to them.
[0086] Laser light incident upon the particle(s) being analyzed may
generate at least one, or perhaps even two, three or more beams of
scattered light or emitted light having specific frequencies,
wavelengths, intensities and perhaps even watts. All or a portion
of the scattered light or even emitted light may be captured by a
detector. A detector may include a photomultiplier tube or the like
detectors.
[0087] A detection system may be used to detecting an amount of
emitted fluorescence from each of the sperm cells in a flow
cytometry system. A detection system or even a sperm cell
fluorescence detector may include a photomultipler tube. In other
embodiments a detection system may include an optical lens
assembly, a photomultipler tube and even some sort of analysis
system such as a computer. An optical lens assembly may collect
emitted fluorescence and transport a collected signal to a
photomultipler tube. The signal detected by a photomultipler tube
may then be analyzed by a computer or the like devices.
[0088] A single or perhaps even a multiple digital or analog
detector(s) may receive all or even a portion of the scattered
light or emission light. Analog or digital processor(s) may convert
the signals detected by the detector(s) into analog current or even
digital information. The information may accurately represent the
identity, frequency, quantity, and even joules of light or EMR
received by the detector.
[0089] In embodiments, a detector may generate a current or digital
signal corresponding to the amount of light quanta hitting the
detector. Certain embodiments of the invention, can provide a one
dimensional detector which summates all light of the specified
wavelength incident upon the detector surface during a specified
period of time. The duration or even a total time of detection may
be as simple as a fully additive collection or even integration
over an entire analysis time of the sample, or it may be a dynamic
data set which may record an emission of light from a biological
object(s) or sample(s) over a time period. That time period may be
as short as the time between two pulses of the pulsed laser light,
or it may be as long as many millions of pulses. It is understood
that such a detector may become two dimensional when the second
dimension of time is considered.
[0090] In other embodiments, a two or three dimensional detector
may comprise a flat panel, a three dimensional matrix of unit cells
or even pixels which can detect an emission light once or more
times and can record or report the interaction specific to that
individual unit cell. The information relevant to the operator of
an apparatus may be a summation or display of the results of many
unit cells. This type of detector may include, but is not limited
to, photographic film, photographic paper, or even a microchip
capable of sending data for imaging on a television screen or
computer screen. It is understood that a two dimensional detector
may become three dimensional, and three dimensional detector may
become dimensional when the additional dimension of time may be
considered.
[0091] A signal generated by a detector can be processed to provide
simple outcomes such as photos. A signal generated by a detector
may even be able to allow analysis of many thousands or even
millions of biological objects in real time with computer graphics
which can give representations to allow a user to modulate or
modify an analysis process in real time. In other embodiments, it
may be desirable to sort with a magnetic detection.
[0092] In embodiments, the present invention may include
quantitatively detecting an amount of emitted fluorescence from
each of the sperm cells. The quantity of the emitted fluorescence
may be detected with a sperm cell fluorescence quantitative
detector. Of course this may include other samples. In sperm cells,
a X chromosome bearing sperm and a Y chromosome bearing sperm may
be distinguished because an X chromosome bearing sperm may emit a
different amount of fluorescence than a Y chromosome bearing sperm.
In other embodiments, if using other samples besides sperm cells,
the present invention may provide distinguishing between a first
population of particles and a second population of particles due to
a difference in an amount of fluorescence emitted from each
population of particles. A distinguishing analysis may be
calculated with a detector.
[0093] Operating system controlled computer(s) or even graphic user
interface controlled computers may use data from an analog or even
digital processor(s). A computer may facilitate direct feedback
control of a laser and even analytical equipment. A computer may
even provide data to support a workstation which may give an
operator(s) of an analytical or separation equipment images that
may relate to a behavior of the system. This may allow control of
the behavior of the system for optimal analysis, quantification,
and even separation of a biological object(s) or even a
sample(s).
[0094] Auxiliary computational, command equipment or even control
equipment may provide local network control of analysis,
quantification and separation apparatus. Control equipment may
communicate in local area networks (LAN) or even wide area networks
(WAN) to provide local or perhaps even distant operators capability
to initiate, monitor, control, troubleshoot, download data or even
instructions, upload data or instructions, terminate, and the like.
Control equipment may allow operation of one, two, three or even
more apparatus.
[0095] Computational subcomponents may correspond to a command and
even a control which may be integrated into a pulsed laser design
and construction. In some embodiments, computational subcomponents
may be independent or even integrated parts of an apparatus and may
reside outside a housing of a pulsed laser.
[0096] In embodiments, the present invention may provide staining a
sperm cell with a fluorescent dye. A stained sperm cell (or in
other embodiments, a stained sample) may be stained with
fluorochrome and in yet other embodiments, may be stained with
Hoechst bisbenzimide H33342 fluorochrome.
[0097] In some instances, a large amount of dye may be used to
stain a sperm cell. Sperm cells contain deoxyribonucleic acid and
deoxyribonucleic acid may have many binding sites that stain (dye)
molecules may bind with. Due to the nature of sperm cells, a
stained sperm cell may have many molecules of a dye attached to
each binding site of a sperm.
[0098] In embodiments, the present invention may include staining a
sperm sample for a reduced staining time. The staining time may
vary due to the type of stain used and even due to the type of
sample used, here sperm. Typically, staining sperm with Hoechst
33324 may take about 40 minutes. under other constant conditions.
Some examples of a reduced staining time may include the following:
[0099] less than about 40 minutes. [0100] less than about 35
minutes; [0101] less than about 30 minutes; [0102] less than about
25 minutes; [0103] less than about 20 minutes; [0104] less than
about 15 minutes; [0105] less than about 10 minutes; and [0106]
less than about 5 minutes. Of course, other stain times are
certainly possible and all should be understood as represented
within the scope of this invention.
[0107] In embodiments, the present invention provides
distinguishing between a X chromosome bearing sperm and a Y
chromosome bearing sperm in a flow cytometer system. A X chromosome
bearing sperm may emit a different fluorescence from said Y
chromosome. For example, a X chromosome bearing sperm may contain
more DNA than a Y chromosome bearing sperm, thus a X chromosome
bearing sperm may bind to more dye. When illuminated, a X
chromosome bearing sperm may emit a greater fluorescence than a Y
chromosome bearing sperm. The difference may provide the ability to
distinguish the two chromosome bearing sperms.
[0108] The present invention may include, in embodiments, minimally
staining sperm cells with a fluorescent dye. A minimum sperm stain
may include allowing less stain to bind to each sperm. For example,
it may take a certain amount of stain to complete attach stain
molecules to each binding site of a sperm cell. It may be an
efficient option if less amount of stain could be used while
maintaining the ability to achieve a desired result, such as the
ability to distinguish between two different cells after radiation.
In embodiments, the present invention may include providing a
percentage of stain. While a percentage of stain may be as low as
10%, other examples may include about 90%, about 80%, about 70% and
about 60%. All stain percentages are understood as included within
the scope of this disclosure.
[0109] A benefit with respect to sorting sperm cells using a pulsed
laser can be a reduction in the amount of stain taken up by sperm
cells during staining. Because stains or dyes, such as Hoechst
33342, bind with DNA within sperm cells, stain has been considered
a factor detrimental to the viability or fertility of sperm cells.
Using a pulsed laser flow sort invention, the amount of stain taken
up by sperm cells during staining can be reduced by 20% over the
amounts used with conventional CW laser cell sorting technology
with similar or better resolution of X-chromosome bearing and
Y-chromosome bearing subpopulations. In certain sperm cell samples
the amount of stain taken up by the sperm cells can be reduced to
as little as 60% of the amount used with the same cells sorted by
conventional CW cell sorting technology.
[0110] Any kind of sample or particle may be used in a flow
cytometry system. A sample may include usable cells, reproductive
cells, haploid cells, sperm cells, delicate sample, non-biological
particles, biological particles, or any kind of cell that can be
used with a flow cytometer system. A useable cell may be a cell
that can be used for further processing or analysis after
completion through a cytometry system. Specifically, in
embodiments, this may include providing a viable cell. Reproductive
cells may include cells that can be used to reproduce an organism
or even a mammal and the like. Haploid cells may include those
cells that have a single set of chromosomes, such as sperm cells. A
delicate sample may include a sample that is fragile or may even be
easily damaged such as reduction in viability. A delicate sample
may have increased sensitivity to certain environments such as the
type of stain, the sorting process and other environments.
[0111] Particles can be non-biological particles such as plastic
beads, biological particles such as diploid cells, haploid sperm
cells, or the like. It is to be understood that particles are not
limited to cells or beads but can also include other non-biological
particles, biological particles, and the like. Particles may
include, without limitation: the individual binding sites or
attachment sites of a molecule on the surface of a cell or other
molecule; large molecules (possibly whether on the surface of a
cell or within a cell) such as proteins, single stranded DNA,
double stranded DNA, mRNA, tRNA, DNA-RNA duplexes, combined protein
nucleic acid structures such as a ribosomes, telomerases or the
like, DNA or RNA polymerases, samino acid synthetase; the active
site of an enzyme such as luciferase, peroxidase, dehydrogenase or
even cytochrome oxidase which may require cofactors such as ATP,
NADH or NADPH; free or bound hydride (H--); or even any structure
biological or non-biological that can be entrained in a fluid
stream and made incident to an illumination beam to generate
scattered light or even emission light.
[0112] In another embodiment that may contribute to efficiency in a
flow cytometry system, the present invention may include subjecting
sperm cells to low power radiation. While the range of power that
may be used with a flow cytometry system may vary, some
possibilities for low power may include: [0113] less than 300
milliwatt; [0114] less than 350 milliwatt; [0115] less than 200
milliwatt; [0116] less than 175 milliwatt; [0117] less than 100
milliwatt; [0118] less than 88 milliwatt; [0119] less than 50
milliwatt; and [0120] less than 25 milliwatt. Again, other powers
of radiation are certainly possible and all should be understood as
represented within the scope of this invention.
[0121] In some examples, a Vanguard Laser may be used. The Vanguard
Laser is manufactured by Spectra-Physics and can emit 80 million
pulses per second (80 MHz). LaserForefront, Spectra-Physics, No. 30
(2001). Each pulse may have line width of about 12 picoseconds, and
a repetition rate of about 10 nanoseconds. This may mean that to an
approximation, during a single repetition of 10 nanoseconds, the
pulsed laser may illuminate a target for about 12 of 10,000
picoseconds or about 0.12% of the total time. In other words, a
sample being illuminated by a pulsed laser may be spending
approximately 99.88% of the time in the dark. This may also mean
that since a pulsed laser may be delivering 350 milliwatts (mW) of
total power, during the short 12 picosecond pulse, an average of
280 Watts may be delivered to a particle. This may be 800 times
more intense than a light from a continuous wave (CW) laser running
at 350 mW. Since reliable sperm sorting can be performed at 150 mW
on a standard CW UV laser, which may represent a factor of 653
fold, it could be hypothesized that it may be possible to run a
pulsed laser at as low as 150/650 or 0.23 mW and still have light
intense enough to illuminate a sperm.
[0122] In embodiments, the present invention may include utilizing
at least one shared resource to process sperm cells. This may help
in efficiency of sperm sorting in flow cytometry systems. A shared
resource may include a computer system, a sheath fluid, an
integrated multiple nozzle device, and the like. In embodiments, a
shared resource may include utilizing one radiation source.
Radiation may be split into at least two beams and each beam may be
directed toward an nozzle and the sample being sorted. In
embodiments, the present invention may include one radiation
emitter and a beam splitter or may even include one intermittingly
punctuated radiation emitter and a beam splitter. A beam splitter
may be any kind of beam splitter as previously discussed.
[0123] As discussed above, the use of refractive, or
semi-reflective splitters provides multiple beams of pulsed laser
light derived from the original source light These beams may have
diminished intensity from the original beam, but may be able to
each be used to analyze separate particles or particle sample(s).
Also discussed above, each beam may be dedicated to an independent
analytical or analytical/separation device (for example, but not
limited to, a sperm sorting flow cytometer or cell sorter). In some
embodiments of the invention, each light beam corresponding to an
independent instrument can be split into two light beams of equal
intensity and one light beam made incident upon the particle to be
analyzed, and the other light beam can provide a reference beam. By
comparing the two beams, the absorption of source light by the
particle may be measured. Another unique and important attribute of
using a single pulsed laser to supply light to dozens or hundreds
or thousands of independent analysis or separating machines may be
that the entire complex of instruments served by the single light
will be using the same light, and to the extent that all machines
are performing identical or highly similar activities, it is
possible to use the data from all machines as internal references
and standards to each other, and by using computers or both which
can give local (LAN) as well as distant (WAN) access to the data,
to allow operators or persons at a distance to monitor the
performance of each machine in real time.
[0124] While multiple nozzles may be integrated into one device,
separate flow cytometers having only one nozzle may be lined up so
that radiation may be directed to or even split between each
nozzle.
[0125] In embodiments, the present invention may include flowing at
least one sheath fluid and sperm cells into at least two nozzles.
By multiplying the number of nozzles operating on a single flow
cytometer, the amount (number of particles) analyzed and sorted per
unit time may be increased. In the case where the operation of the
flow cytometer may be in a production setting representing a
saleable product, multiple nozzle may increase the number of units
produced in a single shift by a single operator, and thus a
reduction in the costs of each unit produced.
[0126] By operating a number of nozzles on the same device, a
controlling instrumentation used on the flow cytometer and
operators of the flow cytometer may use statistical analysis of the
performance (operation data) of a multiple of nozzles and may use
this data for feedback control of single nozzles within the
population of the nozzles being used. By operating a number of
nozzles on the same device, a single light source providing a
multiple of beams (one or more for each nozzle) may reside on the
same mounting as the nozzles and thereby reduce the complexities of
light paths related to nozzles running on individual mountings,
which may be independent of the mounting of the primary
illumination source. By operating a number of nozzles on the same
device illuminated by multiple beams from a single light source
providing the capital, operating, parts, service, and maintenance
costs from a single laser may be distributed across a multiple of
productive sorting nozzles, and, therefore, reduce costs per unit
produced which are allocated to the laser operation.
[0127] FIG. 7 shows multiple nozzles (5) which can provide charged
drops (9). The multiple nozzles and collector (20) may be arranged
so that a number of selected containers may be less than a number
of nozzles. Selected containers may include containers having
collected one specific type of cell, such as all the X chromosome
bearing sperm cells. In this figure, the sorted X chromosome sperm
cells may be represented by the containers (32). Here there are
three selected containers of a selected cell that have been sorted
from four nozzles.
[0128] In other embodiments, the present invention provides
utilizing collected sorted sperm for insemination of female mammals
and may even provide for a mammal produced through use of a sorted
sperm cells produced with a flow cytometer system according to any
of the embodiments as presented in this disclosure.
[0129] In other embodiments, the present invention may include
individually controlling or even compositely controlling at least
two nozzles. Each nozzle may individually adjusted according to a
desired function with an individual nozzle control, or a plurality
of nozzles may be adjusted compositely with a single nozzle control
device that may be connected to each of the nozzles.
[0130] Another way to increase efficiency in a flow cytometry
system, the present invention includes rapidly sorting said sperm
cells. This may be achieved with a rapid sperm sorter or even a
rapid particle sample sorter. Sperm may be sorted at any rate. Such
possibilities for a sort rate may include: [0131] greater than 500
cells per second. [0132] greater than 1000 cells per second; [0133]
greater than 1500 cells per second; [0134] greater than 2000 cells
per second; and [0135] greater than 3000 cells per second. Other
sort rates are certainly possible and all should be understood as
represented within the scope of this invention.
[0136] In embodiments, the present invention may include a particle
sample collector such as a sperm cell collector. A collector may be
multiple containers, a combined collector having individual
container, or any type of collector. For example and as shown in
FIG. 9, a combined collector (63) may include a collector for one
type of particle (32), such as X chromosome bearing sperm
populations, a container for a second type of particle (33) such as
Y chromosome bearing sperm populations, and may even have a third
container (64) to collect those drops which may not have been
charged.
[0137] It may be important in designing illumination beams to
consider that the closer the illumination source (laser) may be to
an analysis point, the less effect any form of movement such as
vibrations may have on the path of the beam. It may be desirable to
provide an system which reduces the distance between and location
of all nozzles to within a very small distance of each other (for
example all within 15 cm), and greatly simplifies and enables the
use of multiple beams from a single laser light source.
[0138] FIG. 9 shows an exploded diagram of components of a flow
cytometry system combined into a parallel construction where a
multiple of nozzles may be operated on a single apparatus. Although
the diagram depicts six nozzles, it is exemplary, such that it
might as easily have only 2 or 3 nozzles, or may have as many as 8
or 10 or 12 or even 24 nozzles side by side.
[0139] A multiple of incoming laser beams or radiation (11), which
in most embodiments could be equal beams derived by splitting from
an original source beam located close to the nozzles, shines onto
an analysis point which is defined by the intersection of the beam
or radiation (11) with a narrow stream of fluid which emits from
the nozzle (5). In some embodiments, the analysis point may be at
the focal point (60) of a reflective parabolic dish (61) which may
reflect emitted light (62) up to the detection surface (58).
Unabsorbed laser light which may not be absorbed may be absorbed by
a heat sink, or it may be measured by an additional detector to
determine an exact real time intensity of the beam. Each nozzle may
be equipped with an oscillator (6) which may provide a force
causing the stream emitting from the nozzle tip or orifice (47) to
break into droplets at defined frequencies such as in the
10,000-100,000 Hz range. Droplets may be charged, and by action of
a magnetic field may be separated. In the case of sorting live
mammalian sperm for the presence of X or Y chromosomes, there can
be 3 streams of droplets: a stream containing primarily X
chromosome bearing sperm, which may by example be collected in one
container (32) on one side of a collector (20), a stream containing
primarily Y chromosome bearing sperm, which may by example be
collected in another container (33) on the other side of a
collector (20), and a stream containing sperm which may be dead and
which may be collected a third container (64) in the middle of a
collector (20). In other embodiments, features such as a detection
surface (58), parabolic dishes (61), collectors (20) and in some
embodiments nozzles (5) and oscillator(s) (6), may be fabricated
into single parts or group subassemblies, which may be sandwiched
together to manifest the actual sorting nozzle architecture.
[0140] In other embodiments of the present invention, detection
surfaces may have diameters (57) similar to the diameter of
microtiter plate wells, which can be about 5-8 millimeters, and can
have distances (59) between two neighboring flow cytometry nozzle
tips which are equal to the distance between two wells, which may
be about 12-18 mm.
[0141] Now, referring primarily to FIG. 10, certain time intervals
and light energy quantities which may be derived from the
particular properties of light provided by a pulsed laser are
shown. The standard lasers used in most flow cytometry and
particularly in sperm sorting have been ion tube continuous wave
(CW) lasers which emit a fairly constant light flux, pulsed lasers
may deliver the same rate of net light. For example, as watts is
defined as joules per second, we may consider the period of 1
second. It may be exemplary that for many applications in flow
cytometry, as many as 10,000-100,000 individual events may be
analyzed in 1 second, so that each event requires illumination
energies of approx 1/10,000-1/100,000 joules.
[0142] In contrast, the pulsed laser may emit the same net light in
regular pulses. In FIG. 15, which represents an arbitrary time
axis, each pulse of laser light (68) can emit a certain energy, and
have a certain illumination pulse duration. When a pulse may
illuminate a particle, a fluorescent emission pulse may occur from
that particle which can be represented by an emission curve (65).
An emission curve can represents a classic exponential decay
function where maximum emission is at the start and the rate of
emission (decay) is along a corresponding half life curve. Based on
some definition of final decay, for example to the point where
emission is 1/1000 of original emission, or about 10 half lives, an
emission pulse duration (67) can be established. There may also be
a period commencing from the final decay point occurring at the end
of the time summated by illumination pulse duration and emission
pulse duration (67) and the beginning of the next illumination
pulse, which can be defined as the resting period. The total sum of
these periods may the period between pulses which may be the
interpulse period and is typically the inverse of the frequency of
the laser.
[0143] Using a detection surface, it may be possible to analyze the
light output emitted from a particle emission pulse and
specifically measuring the summated total of energy from the
emission pulse, which may be an integration of the area (66) under
the decay curve (65). This measurement (70) may be stored as an
analog electrical charge in a charge storage device such as an
appropriate capacitor, of it may be converted to a digital value
(70) which can be stored in a digital memory device. Given that the
emission pulse occurs as a dynamic emission event, which through a
photodiode/amplifier system may be translated in realtime to an
electrical current (or voltage differential), measurements of
current or voltage at multiple points during the particle emission
may allow the derivative of the decay curve to be determined (71).
These can be useful values in statistical analysis of multiple
identical illumination events of the same particle.
[0144] In flow cytometry, which is a broad field in which the
instant invention may be used, particles which are being
illuminated by a laser are commonly flowing in a fluid stream or a
flow cell past a fixed point upon which a laser beam is focused. In
FIG. 11, a rate of flow of particles past the point of illumination
can be a function of the volume flux of the stream, and the
concentration of the particles. An illumination period (72) of time
in which the particle is being illuminated may be determined by the
flow rate and the size of the particle in the direction of the
flow. When the particle may be illuminated by a pulsed laser
generating individual emission pulses (73) which can occur in
interpulse periods much less than the illumination period (72) of
the particle, then a large number of individual emission pulses may
be derived from the particle (74). In contrast, when a particle may
be illuminated during the same period by a continuous wave (CW)
laser, there may be a long emission over the period (75), which can
commonly be detected as a peak profile of current over the period.
A measurement of the emission from particles illuminated by CW
lasers are a single long analog events without natural internal cut
points and so either the entire value may be integrated, or the
event may be sampled at a discreet multiple of times, or segments
of the event are integrated.
[0145] In other embodiments, lasers may be used where the
illumination pulse duration may be much smaller than an interpulse
period which may itself be much shorter than the particle
illumination period (72). For example, when the Vanguard Laser is
used for the sorting of sperm at approximately 25,000 events per
second, the laser which has 80,000,000 illumination pulses per
second will deliver approximately 3000 pulses per event, and about
5-10% or 150-300 pulses occur in the particle illumination period
(72). Also, the pulse duration is about 10 picoseconds (10-11 sec),
the interpulse period is about 10 nanoseconds (10-8 seconds), and
the pulse emission period is less than 1 nanosecond.
[0146] As may be understood from FIG. 12, an illumination pulse may
initiating a sensing routine. The instant invention may use a laser
pulses as an internal clock to the entire analysis system.
Advantages are that each illumination pulse, which is very brief
and very strong, can be used to initiate each clock cycle. Within a
single clock cycle, a computational subroutine may run which uses
the resting period to calculate specific values for each
illumination/emission event, and cache the result prior to the
initiation of the next clock cycle. An analysis of individual
particles could be manifested over a multiple of clock cycles (for
example 150-300), such that statistical analysis of all emission
events mapping to a single particle may occur, and averaged values
related to the measurement of the quantity particle and the
position of the particle may be cached. The period between a
multiple of events, which may be dominated by periods without
emissions, may be used to map the identity and distances and using
the momentary gating criteria to effect the sort. Values of
operating parameters and results within each sort may be cached for
viewing at the 1 minute level, possibly operator status, and
graphic or summations for entire sort runs may be generated. In
FIG. 17, using the example of sperm being sorted using a Vanguard
Laser, the clock cycle may be about 10-8 seconds. Each clock cycle
encompasses three periods. The illumination pulse of 10-11 seconds,
the emission period of 10-9 seconds, and the resting period of
9.times.10-9 seconds. Each clock cycle (77) occurs approximately
300 times in each analysis event of 3.times.10-6 seconds. Time
between analysis events averages 2.times.10-5 seconds. The time
between each analysis and the sort (79) is approximately
5.times.10-2 seconds. Operators will usually want to observe net
historical data over the most recent minute (80) and be able to
view the progress/history of data over the entire sample from start
of sort (81).
[0147] There may be many hierarchical layers of data occurring
dynamically. At the same time, with a number of nozzles all sorting
the same sample at the same time, there are simultaneous events
occurring in each nozzle at each hierarchical layer. As it would be
labor intensive and inefficient for the operator to control each
nozzle, the statistical analysis and algorithmic mapping should
allow the operator to view status, history, and averages of all
nozzles in aggregate and note only nozzles which are not
functioning near the mean of the group. The operator also needs to
use commands to change the sorting, which should effect all nozzles
at one time.
[0148] The data may also be shared between control functions across
multiple nozzles and over time to allow the system to make
automatic adjustments such as: adjusting optical mirror positions
to assure equal laser light in each beam; tracking the performance
of each nozzle to make early identification of nozzle occlusion
events; tracking the performance of each nozzle to identify
differential flow rates and even comparing one or more semen
samples with direct comparisons.
[0149] All of these various calculations, in real time, can be
calibrated very precisely in time, as they may all use the very
high frequency laser clock. Thus, in the parallelized flow
cytometer, a pulsed laser may serve as an important integration
component for all of the data being generated in a multiple of
nozzles.
[0150] Referring primarily to FIG. 13, since it may be desirable to
stain samples just prior to sorting, sorting a sample for a period
of time before staining a second sample which may have been sorted
and repeating this process several times may create samples which
have been stained and sorted at different times, but may be pooled
as a single batch. Aggregated data (82) for each sample may be
compared across a multiple of nozzles and multiple of sorts (83),
for example, from the same ejaculate of a certain bull. Comparisons
of the same bull over multiple days (84) and comparisons between
various bulls (85) can give a history. The data from this history
may reside within the operating system and may be used to assist
operators in choosing staining concentrations or times, or to help
identify ejaculates which are sorting worse than their normal
sorting. Also, if high-throughput resort analysis is available,
predicted sex ratios versus. actual sex ratios may be determined,
and the aggregated sex ratios may be compared to identify settings
and methods which different operators may be using, or to identify
operators who are consistently getting lower results. Also, trends
in the sorting performance may become visible which dictate special
maintenance such as cleaning of optics, replacement of nozzle,
mirror assemblies, or the like.
[0151] Now referring primarily to FIGS. 14 through 18, embodiments
of the invention are shown using a pulsed laser in the context of
flow sorting of sperm cells. Various results from experiments run
at different powers of radiation beams are shown. The different
experiments included 20 mW, 60 mW, 90 mW, 130 mW and 160 mW power
and each power was created using neutral density filters. These
embodiments can provide high-purity sperm sorting for enrichment of
X or Y-chromosome bearing sperm cells which can even be up to 98%
in purity.
[0152] In yet other embodiments, the present invention may provide
collecting at least two populations of sample particles, more
specifically, collecting a sorted population of X chromosome
bearing sperm and collecting a sorted population of Y chromosome
bearing sperm. A collector may be provided to collect each sorted
population. Accordingly, the present invention may include a X
chromosome bearing sperm collector and a Y chromosome sperm
collector. It may be important to sort and collect each population
at a high purity. A high purity sorted population of X chromosome
bearing sperm and said Y chromosome bearing sperm may include a
percentage of purity. Of course, any percentage of purity may exist
and some examples may include: [0153] greater than 85% purity;
[0154] greater than 90% purity; [0155] greater than 95% purity;
[0156] greater than 96% purity; and [0157] greater than 98% purity.
Other percentages of purity are certainly possible and all should
be understood as represented within the scope of this
invention.
[0158] Typical pulsed lasers having characteristics similar to that
set out by Table 1 or Table 2 can be used with the invention.
TABLE-US-00001 TABLE 1 Vanguard 150 mW Output Power Average Output
Power [W] 0.15 UV Beam Size [mm] 1 Energy per Pulse [J] 1.875E-09
Peak Power [W] 234-375 Power per cm{circumflex over ( )}2
[W/cm{circumflex over ( )}2] 2.98E+04
[0159] TABLE-US-00002 TABLE 2 Vanguard 350 mW Output Power Average
Output Power [W] 0.35 UV Beam Size [mm] 1 Energy per Pulse [J]
4.375E-09 Power per Pulse [W] 546.875 Power per cm{circumflex over
( )}2 [W/cm{circumflex over ( )}2] 6.96E+04
[0160] FIGS. 14 through 18 show univariate histograms and a
bivariate dot plots from sorting of Hoechst 33342 stained bovine
sperm cells. Sperm cells sorted were obtained from the same freshly
ejaculated bull sperm diluted to 200.times.10.sup.6 sperm cells per
mL and incubated in Hoechst 33342 at 34.degree. C. for 45 min.
[0161] With respect to the particular histograms and bivariant
plots shown by FIGS. 14 through 19, the event rate (illumination of
the sperm cells as they pass through the pulsed laser beam) was
established at 20,000 events per second. The sort rate (separation
of the sperm cells differentiated by analysis) into subpopulations
was varied from 850-3500 depending on the power used. The results
are also set out by Table 3. TABLE-US-00003 TABLE 3 Pulsed Laser
Power Setting Purity X % Purity Y % 20 mW pulsed 96.5 91.0 60 mW
pulsed 93.5 85.5 90 mW pulsed 96.0 89.5 130 mW pulsed 96.0 91.0 160
mW pulsed 97.0 93.0
[0162] As can be understood from Table 3, using a pulsed laser
sperm cells can be sorted into high purity X-chromosome bearing and
Y-chromosome bearing subpopulations. For each laser power setting
between 20 mW and 160 mW sorted subpopulations had a purity of up
to 97.0% of the correct sex type.
[0163] Now referring primarily to FIG. 19, histograms compare the
resolution of the same sample of sperm cells using conventional CW
flow sorting technology and with an embodiment of the flow sorting
invention utilizing a pulsed laser beam. Importantly, pulsed laser
illumination of stained sperm cells provides superior resolution of
X-chromosome bearing sperm cells and Y-chromosome bearing sperm
cells. This is true even when the pulsed laser beam has a power
significantly lower than the compared to conventional CW flow
sorter technology. Even when the pulsed power is 130 mW, 70 mW or
even 20/150 of the power used in the compared to CW flow sorter
technology. The histograms of the pulsed laser experiments show a
cleaner separation of the two peaks as compared to the continuous
wave experiments.
[0164] In embodiments, the present invention may include providing
a high resolution of a sorted population of sperm cells. A higher
resolution may be indicative of the purity of a sorted population.
While many different resolution values may exist; some examples of
high resolutions may include: [0165] greater than 7.0; [0166]
greater than 7.5; [0167] greater than 8.0; [0168] greater than 8.5;
[0169] greater than 9.0; and [0170] greater than 9.2. Of course,
other resolution values may exist and all are to be understood as
represented within the scope of the invention.
[0171] As discussed above, lower laser power analysis of particles,
and in this particular embodiment of the invention sperm cells,
resolves the long standing problem of having to have a dedicated
laser source for each flow sorter. By reducing the laser power
required, even without achieving any other benefit, multiple flow
cytometers, flow sorters, or cell sorters can be operated using a
single laser source. For example, when sorting is accomplished at
about 20 mW, a single 350 mW pulsed laser can be used to provide
illumination light for as many as 18 separately functioning flow
cytometers or flow sorters used to separate sperm cells on the
basis of bearing an X or Y chromosome.
[0172] Again referring to FIG. 19, another important benefit
provided by a pulsed laser invention in the context of sorting
sperm cells can be increased resolution of X-chromosome bearing and
Y-chromosome bearing subpopulations even when sperm cell samples,
such as those used in this specific example, are stored for long
periods of time at about 5.degree. C., such as 18 hours or longer,
or frozen and thawed prior to staining and analysis, the resolution
of sorted sperm cells can improved with the pulsed laser invention
compared to conventional CW laser technology.
[0173] In embodiments, the present invention may include sorting
sperm cells at a low coincidence rate. Some examples of low
coincidence rates may include: [0174] less than 4400; [0175] less
than 4000; [0176] less than 3700; and [0177] less than 3600. Again,
other low coincidence rates are certainly possible and all should
be understood as represented within the scope of this
invention.
[0178] The present invention may include, in embodiments,
collecting sorted populations of sperm cells at a high collection
rate. A high collection may increase productivity and even
efficiency. Some examples of high collection rates may include:
[0179] greater than 2400 sperm per second; [0180] greater than 2600
sperm per second; [0181] greater than 2900 sperm per second; [0182]
greater than 3000 sperm per second; and [0183] greater than 3100
sperm per second. Other collection rates are possible and all are
meant to be included within the scope of this invention.
[0184] In yet other embodiments, the present invention may include
detecting sperm cells at an event rate of between about 10,000 to
about 60,000 sperm cells per second. Of course, an event rate may
be greater than 10,000 or even lower than 60,000 cells per
second.
[0185] A benefit with respect to sorting sperm cells using the
pulsed laser invention can be higher sorting speeds. When
resolution of a particular sample is increased, the sort rate of
subpopulations of sperm cells to a given purity can be
increased.
[0186] Another benefit with respect to sorting sperm cells using
the pulsed laser invention can be a higher purity sort. When
resolution of a particular sample is increased, the purity of the
subpopulations of sperm cells can be increased.
[0187] A pulsed laser invention may be understood to have
application with respect to any particular particle characteristic
which may be differentiated by change of illumination intensity or
by detectable light emission upon illumination with a pulsed light
beam. While the applicant has provided specific examples of
differentiating the amount of DNA within a cell using the
invention, it to be understood that these examples are illustrative
of how to make and use the invention with regard to the wide
variety of non-biological and biological particles, including, but
not limited to, viral particles, polyploid cells, diploid cells,
haploid cells (such as sperm cells obtained from any species of
mammal such as any type or kind of bovine, ovine, porcine, or
equine sperm cells; or sperm cells obtained from any type or kind
of elk, deer, oxen, buffalo, goats, camels, rabbits, or lama; or
sperm cells obtained from any marine mammal such as whales or
dolphins; or sperm cells obtained from any rare or endangered
species of mammal; or sperm cells obtained from a zoological
species of mammal; or sperm cells obtained from a rare or prize
individual of a species of mammal; or sperm cells obtained from an
individual of a species of mammal that used to produce dairy or
meat products. In embodiments, sperm cells may include any type of
sperm cells such as but not limited to, mammals, bovine sperm
cells, equine sperm cells, porcine sperm cells, ovine sperm cells,
camelid sperm cells, ruminant sperm cells, canine sperm cells and
the like.
[0188] It is understood that the present invention may exist in
unique advantages when combined with other aspects of the various
references incorporated.
[0189] It is also to be understood that these specific examples
provided are not intended to limit the variety of applications to
which the invention may be used, but rather are intended to be
illustrative how to make and use the numerous embodiments of the
invention for application with analytical devices such as flow
cytometers, cell sorters, microarray analyzers, imaging
microscopes, or microimaging equipment, which may easily be built
to contain two or more, and perhaps thousands or even millions
parallel channels for analysis, and in such that each of these
separate channels is capable to perform the identical or very
similar function to a single flow cytometry sorting nozzle, they
may be considered "machines", and it is understood that even a very
small device which could be held in a persons hand, may contain
many hundreds or many thousands of such "machines" and only be able
to function if the use of illumination light is similar or
identical to the inventions described herein.
EXAMPLE 1
[0190] Purified fixed bull sperm heads (also described as bull
sperm nuclei), stained in standard conditions with DNA binding
stain Hoechst 33342, are used as a performance standard to
calibrate a sperm sorting flow cytometer prior to the sorting of
live sperm. A pulsed laser (Spectrophysics VNGD350-HMD355)
delivering 300 mW of energy at 355 nm and 80 MHz illuminates the
sample analysis stream in a flow cytometer operating at standard
settings and provides the histogram plot shown in Fig. Ex 1. This
demonstrates that a standard sperm sorting flow cytometer equipped
with the pulsed laser is able to resolve bull sperm nuclei into
X-chromosome bearing and Y-chromosome bearing populations using
standard conditions.
EXAMPLE 2
[0191] A sample of live bull sperm is stained in standard
conditions with DNA binding stain Hoechst 33342. A pulsed laser
(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355
nm and 80 MHz illuminates the sample analysis stream in a flow
cytometer operating at standard settings sorting said sperm and
provides the histogram plot shown in Fig Ex 2. This demonstrates
that a standard sperm sorting flow cytometer equipped with the
pulsed laser is able to resolve live sperm into X-chromosome
bearing and Y-chromosome bearing populations under standard
conditions.
[0192] The above sample is sorted for collection of X-chromosome
bearing sperm, and the sort collection rate is 3800 live
X-chromosome bearing sperm second. A resort analysis of the sample
prepared in said manner measures the purity of said sorted sample
to be 95%. This demonstrates that a standard sperm sorting flow
cytometer equipped with the pulsed laser is able to enrich the
content of a sperm population from one in which approximately 50%
of the sperm are X-chromosome bearing sperm, to one in which 95% of
the sperm are X-chromosome bearing sperm.
[0193] Said sorted sperm above are further processed by standard
methods for packaging into artificial insemination straws, are
cryopreserved by the standard freezing method, and are thawed for
analysis of motility of the sperm. Percent motility at various
points in the procedure is determined to be: After stain 80%, after
sorting 70%, after cooling 65%, after freezing and thawing at 0
minutes 45%, 30 minutes after thawing 45%, 120 minutes after
thawing 35%. This demonstrates that a standard sperm sorting flow
cytometer equipped with the pulsed laser is able to enrich live
stained sperm samples which appear normal in respect to the sperm
motility.
EXAMPLE 3
[0194] A sample of live bull sperm is stained in standard
conditions with DNA binding stain Hoechst 33342. A pulsed laser
(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355
nm and 80 MHz is equipped with beam splitters and neutral density
filters, in five separate conditions, to provide illumination
energy beam levels of 160 mW (53% of beam power), 130 mW (43% of
beam power), 90 mW (30% of beam power), 60 mW (20% of beam power),
and 20 mW (6.6% of beam power), respectively, to illuminate the
sample analysis stream of a sperm sorting flow cytometer operating
at standard settings sorting said stained sperm and providing the 5
histogram plots shown in Fig Ex 3. This demonstrates that a
standard sperm sorting flow cytometer equipped with the pulsed
laser is able to clearly resolve live sperm with energies as low as
60 mW (20% of the beam).
[0195] The above samples are sorted at each of the 5 beam energy
settings for collection of X-chromosome bearing sperm and
Y-chromosome bearing sperm in separate fractions, and the sort
collection rate is 850-3500 X-chromosome bearing or Y-chromosome
bearing sperm second, depending on the power used, with lower
powers associated with lower sort collection rates. A resort
analysis of the samples prepared in said manner measures the purity
of said sorted samples as shown in Table Ex 3. This demonstrates
that a standard sperm sorting flow cytometer equipped with the
pulsed laser delivering beam energies in the range of 20 mW to 160
mW is consistently able to enrich the content of a sperm population
from one in which approximately 50% of the sperm are X-chromosome
bearing sperm, to one in which 95% or higher of the sperm are
X-chromosome bearing sperm, and simultaneously to one in which 90%
or higher of the sperm are Y-chromosome bearing sperm.
TABLE-US-00004 TABLE 4 Beam Energy (Pulsed) % Purity - X % Purity -
Y 20 mW 96.5 91.0 60 mW 93.5 85.5 90 mW 96.0 89.5 130 mW 96.0 91.0
160 mW 97.0 93.0
EXAMPLE 4
[0196] A sample of live bull sperm is stained in standard
conditions with DNA binding stain Hoechst 33342. A pulsed laser
(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355
nm and 80 MHz is equipped with beam splitters and neutral density
filters, in two separate conditions, to provide illumination energy
beam levels of 130 mW (43% of beam power) and 70 mW (23% of beam
power), respectively, to the sample analysis stream of a flow
cytometer operating at standard settings sorting said stained
sperm. As comparison, the same sample is analyzed on two identical
but different flow cytometers operating at standard settings and
equipped with a CW (continuous wave) lasers delivering 150 mW in
both cases. This demonstrates that even with lower beam energies a
standard sperm sorting flow cytometer equipped with the pulsed
laser provides superior resolution capability when compared to a
same standard sperm sorting flow cytometer equipped with a standard
CW laser.
EXAMPLE 5
[0197] A sample of live bull sperm is stained in standard
conditions with DNA binding stain Hoechst 33342 with the standard
concentration of Hoechst 33342 being defined as 100% level of stain
(control). Two additional samples are prepared which are identical
except that they are stained with 80% or 60% of the amount of
Hoechst 33342 stain, respectively. A pulsed laser (Spectrophysics
VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is
equipped with beam splitters and neutral density filters, in two
separate conditions, to provide illumination energy beam levels of
150 mW (50% of beam power) and 90 mW (30% of beam power),
respectively, to the sample analysis stream of a flow cytometer
operating at standard settings sorting said stained sperm with 3
different concentrations of stain used. The resolution between
X-chromosome bearing and Y-chromosome bearing sperm for these 6
conditions are provided in the 6 histogram plots shown in Fig Ex 5.
This demonstrates that lesser amounts of Hoechst 33342 stain may be
used to prepare sperm samples for sorting on a standard sperm
sorting flow cytometer, if higher pulsed beam energies are also
used.
EXAMPLE 6
[0198] Purified fixed bull sperm heads (also described as bull
sperm nuclei), stained in standard conditions with DNA binding
stain Hoechst 33342, are used as a performance standard to
calibrate a sperm sorting flow cytometer prior to the sorting of
live sperm. A pulsed laser (Spectrophysics VNGD350-HMD355)
delivering 300 mW of energy at 355 nm and 80 MHz and equipped with
a beam splitter to provide an illumination energy beam level of 150
mW (50% of beam power) illuminates the sample analysis stream of a
flow cytometer operating at standard settings. Said stained nuclei
are analyzed at 20,000 events/second (a rate comparable to the rate
used in live bull sperm analysis), as well as at 59,000
events/second. The resolution between X-chromosome bearing and
Y-chromosome bearing bull sperm nuclei for these 2 event rate
conditions are provided in the 2 histogram plots shown in Fig Ex 6.
This demonstrates, that for ideal particles such as nuclei
standards, the event rates of analysis may be increased as much as
3-fold with only modest loss in the resolution between X-chromosome
bearing and Y-chromosome bearing bull sperm nuclei.
EXAMPLE 7
[0199] Samples of live bull sperm from 4 different bulls were
stained in standard conditions with DNA binding stain Hoechst
33342. A pulsed laser (Spectrophysics VNGD350-HMD355) delivering
300 mW of energy at 355 nm and 80 MHz is equipped with beam
splitters and neutral density filters, in two separate conditions,
to provide illumination energy beam levels of 300 mW (100% of beam
power) and 150 mW (50% of beam power), respectively, to the sample
analysis stream of a flow cytometer operating at standard settings
sorting said stained sperm samples. As comparison, the same samples
are sorted on an identical but different flow cytometer operating
at standard settings and equipped with a CW (continuous wave) laser
delivering 150 mW of energy in the illumination beam. The samples
are bulk sorted, which means both X-chromosome bearing and
Y-chromosome bearing sperm fractions are pooled. The sorted sperm
samples are cryopreserved using standard procedures and the percent
of post thaw sperm motilities, as well as the percent of live and
dead using PI staining with flow cytometry analysis are scored. The
averages for all 4 bulls with the 3 different illumination
conditions are shown in Table 5. This demonstrates without
statistical significance that all three conditions of illumination
yield similar numbers of intact viable sperm after sorting.
TABLE-US-00005 TABLE 5 % Motility at % Motility at % Live % Live 0
min 90 min at 0 min at 90 min Laser (mW) post thaw post thaw post
thaw post thaw CW (150 mW) 50.0 42.5 43.5 40.6 Pulsed (150 mW) 46.3
42.5 40.0 37.9 Pulsed (300 mW) 48.1 36.3 40.3 37.2
EXAMPLE 8
[0200] Samples of live bull sperm from 5 different bulls are
stained in standard conditions with DNA binding stain Hoechst
33342, with the standard concentration of Hoechst 33342 being
defined as 100% level of stain (control). Two additional samples
from the same 5 bulls are prepared which are identical except that
they are stained with 80% or 60% of the amount of Hoechst 33342
stain, respectively.
[0201] A pulsed laser (Spectrophysics VNGD350-HMD355) delivering
300 mW of energy at 355 nm and 80 MHz is equipped with beam
splitters and neutral density filters, in two separate conditions,
to provide illumination energy beam levels of 150 mW (50% of beam
power) and 90 mW (30% of beam power), respectively, to the sample
analysis stream of a flow cytometer operating at standard settings
sorting said stained sperm samples. As comparison, the same samples
are sorted on an identical but different flow cytometer operating
at standard settings and equipped with a CW (continuous wave) laser
delivering 150 mW of energy in the illumination beam.
[0202] For the sorting procedures on all these samples, the average
values for resolution (higher values are better), the coincidence
rates (lower values are better), the sort collection rates (higher
values are better) are compared and shown in Table 6. This
demonstrates that sorting efficiencies in all conditions tested
with the pulsed laser were equal to or better than the sorting
efficiencies achieved using the standard CW laser. TABLE-US-00006
TABLE 6 Co-incidence Stain(%)/Laser (mW) Resolution rate Sort Rate
100/150 pulsed 8.0 3570 3160 100/90 pulsed 9.3 3560 2610 80/150
pulsed 8.2 3600 3160 80/90 pulsed 9.6 3500 2480 60/150 pulsed 8.6
3600 2940 60/90 pulsed 9.8 3520 2450 100/150 CW 7.6 4380 2720
[0203] The same samples are bulk sorted, which means both
X-chromosome bearing and Y-chromosome bearing sperm fractions are
pooled. The sorted sperm samples are cryopreserved using standard
procedures and the percent of post thaw motilities, as well as the
percent of live/dead using PI staining with flow cytometry analysis
are scored. The averages for all 5 bulls with the 7 different stain
and illumination conditions are shown in Table 7. This demonstrates
that sperm viability and live counts in all conditions tested with
the pulsed laser were equal to or better than the sperm viability
and live counts achieved using the standard CW laser and standard
stain. TABLE-US-00007 TABLE 7 % Motility at % Motility at % Live %
Live Stain(%)/ 0 min 120 min at 30 min at 120 min Laser (mW) post
thaw post thaw post thaw post thaw 100/150 pulsed 43.3 34.0 36.8
32.4 100/90 pulsed 42.8 33.5 35.3 33.2 80/150 pulsed 42.8 33.8 35.1
35.1 80/90 pulsed 42.0 35.3 35.2 29.7 60/150 pulsed 40.8 31.0 35.8
35.1 60/90 pulsed 41.0 32.8 34.4 33.7 100/150 CW 39.8 33.3 33.4
28.6
EXAMPLE 9
[0204] Samples of live bull sperm from 5 different bulls and 2-6
replicates were stained in standard conditions with DNA binding
stain Hoechst 33342 (80%) and bulk sorted under standard conditions
in a sperm sorting flow cytometer at event rates of 23,000
sperm/second. A pulsed laser (Spectrophysics VNGD350-HMD355)
delivering 300 mW of energy at 355 nm and 80 MHz is equipped with
beam splitter to provide illumination energy beam level of 150 mW
(50% of beam power) to the sample analysis stream of a flow
cytometer operating at standard settings sorting said stained sperm
samples.
[0205] As a control comparison, same samples of live bull sperm
from 5 different bulls and 2-6 replicates were stained in standard
conditions with DNA binding stain Hoechst 33342 (100%) and these
samples were sorted on an identical but different flow cytometer
operating at standard settings and equipped with a CW (continuous
wave) laser delivering 150 mW of energy in the illumination
beam.
[0206] Said various sperm samples were used at concentrations of
200,000 sperm/ml or 1 million sperm/ml to inseminate matured bovine
oocytes in standard procedures of in-vitro fertilization (IVF).
Cleavage and 2 cell rates at 2.75 days post-insemination,
blastocyst development rates at 7.75 days post-insemination, total
cell numbers in blastocyst and the blastocyst quality at 7.75 days
were measured. The average results for 587 oocytes inseminated with
sorted sperm prepared from the system equipped with the pulsed
laser, and for 558 oocytes inseminated with sorted sperm prepared
from the system equipped with the CW laser are shown in Table 8.
Note: lower numbers for blastocyst quality are better. This
demonstrates that sperm prepared by a standard sperm sorting flow
cytometer equipped with the pulsed laser is capable of fertilizing
oocytes in standard IVF procedures and exhibits similar cleavage
and blastocyst rates, with the mean quality of blastocysts being
slightly better when inseminated with sperm sorted using the
standard flow cytometer equipped with the pulsed laser.
TABLE-US-00008 TABLE 8 % % % quality of cell counts in Laser (mW)
Cleaved 2 cell blastocyst blastocyst blastocyst CW (150 mW) 50.7
27.3 6.7 2.7 131.8 Pulsed (150 mW) 49.7 29.5 5.2 2.1 136.2
EXAMPLE 10
[0207] Samples of live bull sperm from 3 different bulls, on
multiple days, were stained in standard conditions with DNA binding
stain Hoechst 33342 (100%) and bulk sorted under standard
conditions in a sperm sorting flow cytometer at event rates of
20-23,000 sperm/second. A pulsed laser (Spectrophysics
VNGD350-HMD355) delivering 300 mW of energy at 355 nm and 80 MHz is
equipped with a beam splitter to provide illumination energy beam
level of 150 mW (50% of beam power) to the sample analysis stream
of a flow cytometer operating at standard settings sorting said
stained sperm samples.
[0208] As a control comparison, same samples of live bull sperm
from the same 3 different bulls on same days, were stained in
standard conditions with DNA binding stain Hoechst 33342 (100%) and
these samples were sorted on an identical but different flow
cytometer operating at standard settings and equipped with a CW
(continuous wave) laser delivering 150 mW of energy in the
illumination beam.
[0209] Said various sperm samples were used in amounts of 2 million
sperm per cyropreserved artificial insemination straw containing
0.25 ml of fluid.
[0210] Control straws containing 10 million unsorted sperm, and
control straws containing 2 million X enriched sperm sorted using a
sperm sorting flow cytometer equipped with a the standard CW laser
were used.
[0211] In a heterospermic analysis, X-fractions from CW laser sorts
were mixed in equal sperm numbers with Y-fractions from pulsed
laser sorts to create the #1 comparision. Y-fractions from CW laser
sorts were mixed in equal sperm numbers with X-fractions from
pulsed laser sorts to create the #2 comparison. Identification of
sex in fetuses at 60 days was used as a marker to assign the sex
outcome, and accordingly, the likely condition (which laser) can be
attributed to successful fertilization. The heterospermic method is
particularly useful, as all other factors than sorting procedure
are internally controlled in each insemination.
[0212] Holstein heifers weighing approximately 750 pounds were
synchronized using CIDR/Lutalase. Thereafter observed (AM or PM)
for standing heat and were inseminated at 12-24 hours after heat
observation. Using 2 inseminators, and a single deep uterine
insemination treatment, with 5 test groups spaced approximately one
month apart, at a single farm, pregnancy rates and sex of fetus
were determined at 60 days post insemination using ultrasonograhy.
The results shown in Table 9 demonstrate that the sperm sorted with
a standard sperm sorting flow cytometer equipped with a pulsed
laser give essentially identical pregnancy rates as sperm sorted
using a standard sperm sorting flow cytometer equipped with the
standard CW laser. TABLE-US-00009 TABLE 9 % Conception
Preganancies/ Sperm dose type Rate Inseminations Unsexed control
containing 10 million 62.5 55/88 total sperm X-sexed control
containing 2 million 56.4 101/179 total sperm Heterospermic #1
containing 2 million 50.0 45/90 total sperm Heterospermic #2
containing 2 million 58.4 52/89 total sperm Pregancies attributed
to CW laser 49.50% 48/97 Pregancies attributed to pulsed laser
50.50% 49/97
EXAMPLE 11
[0213] A sample of live dolphin sperm was collected at poolside,
shipped via air freight, and stained with Hoechst 33342
approximately 6 hours after collection. The sorting efficiencies
for the single stained sample were then tested on two identical Mo
Flo SX sperm sorters, in one case equipped with a pulsed laser
(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355
nm and 80 MHz equipped with a beam splitter to provide illumination
energy beam level of 150 mW (50% of beam power), and the second
case with an Innova 90-6 (CW--continuous wave) laser delivering 150
mW of beam energy. The dolphin ejaculate was stained 3 times, and
in each case sorted for approximately 2 hours.
[0214] Using the CW laser, with sorter event rates at 30,000/sec an
average co-incidence rate of 6430/sec was observed, X-chromosome
bearing sperm were collected at an average rate of 3450/sec and a
total of 72 million sperm were collected in 7 hours for an average
recovery of 10.3 million sperm per hour.
[0215] Using the pulsed laser, with sorter event rates at
30,000/sec an average co-incidence rate of 5400/sec was observed,
X-chromosome bearing sperm were collected at an average rate of
3930/sec and a total of 79.5 million sperm were collected in 6.33
hours for an average recovery of 12.6 million sperm per hour.
[0216] The recovered sperm from both samples has X purities of
>95% and post-thaw motility of >50%.
EXAMPLE 12
[0217] A sample of live canine sperm was collected from a common
dog housed at a kennel and stained about 3 hours later with a
non-optimized quantity of Hoechst 33342. The stained sperm were
analyzed by a standard sperm sorter equipped with a pulsed laser
(Spectrophysics VNGD350-HMD355) delivering 300 mW of energy at 355
nm and 80 MHz equipped with a beam splitter to provide illumination
energy beam level of 150 mW (50% of beam power). Approximately 43%
of the sperm were correctly oriented. From the correctly oriented
stained canine sperm, approximately 32% were collected as
X-chromosome bearing sperm, and approximately 36% were collected as
Y-chromosome bearing sperm. Visual inspection by microscope showed
high numbers of canine sperm in both samples to be motile.
EXAMPLE 13
[0218] The standard CW laser uses a cathode tube which requires an
average input of 12 KW of electrical power, and a large volume of
cooling water, or a chiller with a load of approximately 15 KW. The
pulsed laser (Spectrophysics VNGD350-HMD355 delivering 300 mW of
energy at 355 run and 80 MHz) requires approximately 500 watts (0.5
KW).
[0219] The standard CW laser also requires replacement of the
cathode tube after approximately 5000 hours of use, at a
replacement cost of about $12,000, whereas the VNG pulsed laser is
expected to have 30,000+hours of operation before refurbishment of
head element at similar costs.
[0220] A commercial operation using the sperm sorting flow
cytometers to sort sperm for production of artificial insemination
straws, running 24 hours per day, year-round, may be expected to
operate lasers for 8,640 hours per year.
[0221] Electric utility and water rates in Fort Collins quoted for
the year 2004 were used to calculate the operating costs of the
standard CW laser, in the first case cooled by utility water and in
the second case cooled using electric powered chiller. The pulsed
laser requires no cooling. The comparative costs are shown in Table
10. This demonstrates that the pulsed laser has significant
benefits in reducing the costs of operation of a sperm sorting flow
cytometer. TABLE-US-00010 TABLE 10 CW laser CW laser with water
with electric Pulsed Cost component cooling chiller cooling Laser
Electrical Charges $4,389 $9,828 $183 Water Charges $6,483 $0 $0
Laser tube or rebuild $20,736 $20,736 $3,456 TOTAL (1 year) $31,608
$30,564 $3,639
[0222] As can be easily understood from the foregoing, the basic
concepts of the present invention may be embodied in a variety of
ways. It involves both sorting techniques as well as devices to
accomplish the appropriate sorting system. In this application, the
sorting techniques are disclosed as part of the results shown to be
achieved by the various devices described and as steps which are
inherent to utilization. They are simply the natural result of
utilizing the devices as intended and described. In addition, while
some devices are disclosed, it should be understood that these not
only accomplish certain methods but also can be varied in a number
of ways. Importantly, as to all of the foregoing, all of these
facets should be understood to be encompassed by this
disclosure.
[0223] The discussion included in this application is intended to
serve as a basic description. The reader should be aware that the
specific discussion may not explicitly describe all embodiments
possible; many alternatives are implicit. It also may not fully
explain the generic nature of the invention and may not explicitly
show how each feature or element can actually be representative of
a broader function or of a great variety of alternative or
equivalent elements. Again, these are implicitly included in this
disclosure. Where the invention is described in device-oriented
terminology, each element of the device implicitly performs a
function. Apparatus claims may not only be included for the device
described, but also method or process claims may be included to
address the functions the invention and each element performs.
Neither the description nor the terminology is intended to limit
the scope of the claims included in this or in any subsequent
patent application.
[0224] It should also be understood that a variety of changes may
be made without departing from the essence of the invention. Such
changes are also implicitly included in the description. They still
fall within the scope of this invention. A broad disclosure
encompassing both the explicit embodiment(s) shown, the great
variety of implicit alternative embodiments, and the broad methods
or processes and the like are encompassed by this disclosure and
may be relied upon when drafting the claims for the full patent
application. This patent application seeks examination of as broad
a base of claims as deemed within the applicant's right and is
designed to yield a patent covering numerous aspects of the
invention both independently and as an overall system.
[0225] Further, each of the various elements of the invention and
claims may also be achieved in a variety of manners. This
disclosure should be understood to encompass each such variation,
be it a variation of an embodiment of any apparatus embodiment, a
method or process embodiment, or even merely a variation of any
element of these. Particularly, it should be understood that as the
disclosure relates to elements of the invention, the words for each
element may be expressed by equivalent apparatus terms or method
terms--even if only the function or result is the same. Such
equivalent, broader, or even more generic terms should be
considered to be encompassed in the description of each element or
action. Such terms can be substituted where desired to make
explicit the implicitly broad coverage to which this invention is
entitled. As but one example, it should be understood that all
actions may be expressed as a means for taking that action or as an
element which causes that action. Similarly, each physical element
disclosed should be understood to encompass a disclosure of the
action which that physical element facilitates. Regarding this last
aspect, as but one example, the disclosure of a "sorter" should be
understood to encompass disclosure of the act of "sorting"--whether
explicitly discussed or not--and, conversely, were there
effectively disclosure of the act of "sorting", such a disclosure
should be understood to encompass disclosure of a "sorter" and even
a "means for sorting" Such changes and alternative terms are to be
understood to be explicitly included in the description.
[0226] Any acts of law, statutes, regulations, or rules mentioned
in this application for patent; or patents, publications, or other
references mentioned in this application for patent are hereby
incorporated by reference. In addition, as to each term used it
should be understood that unless its utilization in this
application is inconsistent with such interpretation, common
dictionary definitions should be understood as incorporated for
each term and all definitions, alternative terms, and synonyms such
as contained in the Random House Webster's Unabridged Dictionary,
second edition are hereby incorporated by reference. Finally, all
references listed herein and in the table of references as listed
below or other information statement filed with the application are
hereby appended and hereby incorporated by reference, however, as
to each of the above, to the extent that such information or
statements incorporated by reference might be considered
inconsistent with the patenting of this/these invention(s) such
statements are expressly not to be considered as made by the
applicant(s). TABLE-US-00011 I. U.S. PATENT DOCUMENTS FILING
DOCUMENT NO DATE NAME CLASS SUBCLASS DATE 2002/0113965 A1 Aug. 22,
2002 Roche et al. 356 339 Oct. 02, 2001 2002/0141902 A1 Oct. 03,
2002 Ozasa et al. 422 85.09 Mar. 27, 2002 2002/0186375 A1 Dec. 12,
2002 Asbury et al. 356 440 May 01, 2001 2003/0098421 A1 May 29,
2003 Ho 250 458.1 Nov. 27, 2001 2003/0207461 A1 Nov. 06, 2003 Bell
et al. 436 172 Nov. 14, 2001 2003/0209059 A1 Nov. 13, 2003 Kawano
et al. 73 53.01 Mar. 28, 2003 2004/0005582 A1 Jan. 08, 2004
Shipwash 435 6 Dec. 19, 2002 3,893,766 Jul. 08, 1975 Hogg 356 36
Jun. 14, 1973 4,362,246 Dec. 07, 1982 Adair 209 3.3 Jun. 14, 1980
4,660,971 Apr. 28, 1987 Sage et al. 356 39 May 03, 1984 4,988,619
Jan. 29, 1991 Pinkel 435 30 Nov. 30, 1987 5,088,816 Feb. 18, 1992
Tomioka et. al 356 39 Mar. 06, 1990 5,135,759 Aug. 04, 1992 Johnson
424 561 Apr. 26, 1991 5,315,122 May 24, 1994 Pinsky, et al. 250
461.2 5,371,585 Dec. 06, 1994 Morgan et al. 356 246 Nov. 10, 1992
5,439,362 Aug. 08, 1995 Spaulding 424 185.1 Jul. 25, 1994 5,466,572
Nov. 14, 1995 Sasaki et al. 435 2 Apr. 25, 1994 5,483,469 Jan. 09,
1996 Van den Engh et al. 364 555 Aug. 02, 1993 5,596,401 Jan. 21,
1997 Kusuzawa 356 23 Sep. 14, 1994 5,602,039 Feb. 11, 1997 Van den
Engh 436 164 Oct. 14, 1994 5,602,349 Feb. 11, 1997 Van den Engh 73
864.85 Oct. 14, 1994 5,660,997 Aug. 26, 1997 Spaulding 435 7.21
Jun. 07, 1995 5,690,895 Nov. 25, 1997 Matsumoto et al. 422 73 Dec.
06, 1996 5,700,692 Dec. 23, 1997 Sweet 436 50 Sep. 27, 1994
5,726,364 Mar. 10, 1998 Van den Engh 73 864.85 Feb. 10, 1997
5,793,485 Aug. 11, 1998 Gourley 356 318 Jan. 13, 1997 5,895,922
Apr. 20, 1999 Ho 250 491.2 May 23, 1997 5,985,216 Nov. 16, 1999
Rens, et al. 422 73 Jul. 24, 1997 6,149,867 Nov. 21, 2000 Siedel,
et al. 422 73 Dec. 31, 1997 6,177,277 B1 Jan. 23, 2001 Soini 436 63
Jan. 03, 1996 6,263,745 Jul. 24, 2001 Buchanan, et al. Dec. 03,
1999 6,357,307 Mar. 19, 2002 Buchanan, et al. 73 865.5 Jul. 20,
2001 6,411835 B1 Jun. 25, 2002 Modell et al. 600 407 Feb. 02, 1999
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Mar. 04, 2003 Karsten, et al. 250 459.1 Jun. 01, 2001 6,534,308 B1
Mar. 18, 2003 Palsson et al. 435 288.7 Nov. 30, 1999 6,537,829 Mar.
25, 2003 Zarling, et al. 436 514 Dec. 01, 1999 6,577,387 B2 Jun.
10, 2003 Ross, III et al. 356 124 Dec. 29, 2000 6,590,911 B1 Jul.
08, 2003 Spinelli et al 372 22 Jun. 02, 2000 6,604,435 Mar. 13,
2002 Buchanan, et al. Aug. 12, 2003 6,618,679 B2 Sep. 09, 2003
Loehrlein et al. 702 20 Jan. 27, 2001 6,642,018 B1 Nov. 04, 2003
Koller et al. 435 40.5 Mar. 13, 2000 6,667,830 B1 Dec. 23, 2003
Iketaki et al. 359 368 Apr. 09, 1999 6,671,044 B2 Dec. 30, 2003
Ortyn et al. 356 326 Nov. 16, 2001 6,673,095 B2 Jan. 06, 2004
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[0227] TABLE-US-00012 II. FOREIGN PATENT DOCUMENTS DOCUMENT NO DATE
COUNTRY EP 0 288 029 20.04.88 Europe WO 96/12171 Apr. 25, 1996 PCT
WO 98/34094 Aug. 06, 1998 PCT WO 99/05504 Jul. 24, 1998 PCT WO
99/33956 Jul. 08, 1999 PCT WO 01/40765 Jun. 07, 2001 PCT WO
01/40765 Jul. 06, 2001 PCT WO 01/85913 Nov. 15, 2001 PCT
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[0229] In drafting any claims at any time whether in this
application or in any subsequent application, it should also be
understood that the applicant has intended to capture as full and
broad a scope of coverage as legally available. To the extent that
insubstantial substitutes are made, to the extent that the
applicant did not in fact draft any claim so as to literally
encompass any particular embodiment; and to the extent otherwise
applicable, the applicant should not be understood to have in any
way intended to or actually relinquished such coverage as the
applicant simply may not have been able to anticipate all
eventualities; one skilled in the art, should not be reasonably
expected to have drafted a claim that would have literally
encompassed such alternative embodiments.
[0230] Further, if or when used, the use of the transitional phrase
"comprising" is used to maintain the "open-end" claims herein,
according to traditional claim interpretation. Thus, unless the
context requires otherwise, it should be understood that the term
"comprise" or variations such as "comprises" or "comprising", are
intended to imply the inclusion of a stated element or step or
group of elements or steps but not the exclusion of any other
element or step or group of elements or steps. Such terms should be
interpreted in their most expansive form so as to afford the
applicant the broadest coverage legally permissible.
* * * * *
References