U.S. patent application number 14/581536 was filed with the patent office on 2015-06-04 for apparatus and method for producing high purity x-chromosome bearing and/or y-chromosome bearing populations of spermatozoa.
This patent application is currently assigned to XY, LLC. The applicant listed for this patent is XY, LLC. Invention is credited to Kenneth M. Evans, Erik B. van Munster.
Application Number | 20150152497 14/581536 |
Document ID | / |
Family ID | 27394507 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150152497 |
Kind Code |
A1 |
Evans; Kenneth M. ; et
al. |
June 4, 2015 |
APPARATUS AND METHOD FOR PRODUCING HIGH PURITY X-CHROMOSOME BEARING
AND/OR Y-CHROMOSOME BEARING POPULATIONS OF SPERMATOZOA
Abstract
A flow cytometer apparatus and method for differentiating
X-chromosome bearing sperm and Y-chromosome bearing sperm having
differing amounts of a light emission material coupled to nuclear
their DNA. The apparatus may include an irradiation source which
generates an irradiation beam responsive to the light emission
material coupled to stained sperm cells, optics to focus the
irradiation beam to a beam pattern having a height about equal to
the length of said sperm cells along the longitudinal axis to about
three times the length of said sperm cells along the longitudinal
axis, and a detector responsive to said light emitted from said
light emission material in response to the irradiation beam. The
method may include the steps of establishing a fluid stream,
producing an irradiation beam directed at the fluid stream, shaping
the irradiation beam to have a beam height between about equal to
the length of said sperm cells along the longitudinal axis and
about three times the length of said sperm cells along the
longitudinal axis at the fluid stream, and detecting emissions from
sperm cells passing through the beam pattern.
Inventors: |
Evans; Kenneth M.; (College
Station, TX) ; van Munster; Erik B.; (Amsterdam,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
XY, LLC |
Navasota |
TX |
US |
|
|
Assignee: |
XY, LLC
Navasota
TX
|
Family ID: |
27394507 |
Appl. No.: |
14/581536 |
Filed: |
December 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13752057 |
Jan 28, 2013 |
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14581536 |
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12113684 |
May 1, 2008 |
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13752057 |
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10275770 |
Feb 7, 2003 |
7371517 |
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PCT/US01/15150 |
May 9, 2001 |
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12113684 |
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60267571 |
Feb 10, 2001 |
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60239752 |
Oct 12, 2000 |
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60203089 |
May 9, 2000 |
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Current U.S.
Class: |
435/6.1 ;
435/287.2 |
Current CPC
Class: |
B03B 9/00 20130101; C12N
5/061 20130101; C12N 5/0612 20130101; C12Q 1/6879 20130101; G01F
17/00 20130101; G01N 15/14 20130101; G01N 21/17 20130101; G01N
15/147 20130101; G01N 2015/149 20130101; A61D 19/00 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 15/14 20060101 G01N015/14 |
Claims
1. A flow cytometer for differentiating sperm cells, wherein the
sperm cells have an associated light emission material coupled to
nuclear DNA and wherein the light emissions material stains
X-chromosome bearing sperm and Y-chromosome bearing sperm in
differing amounts comprising: a) an irradiation source which
generates an irradiation beam responsive to the light emission
material coupled to stained sperm cells in a fluid stream; b)
optics to focus the irradiation beam, wherein said optics focus a
beam pattern having a height of about equal to the length of said
sperm cells along the longitudinal axis to about three times the
length of said sperm cells along the longitudinal axis; and c) a
detector responsive to said light emitted from said light emission
material in response to the irradiation beam.
2. The flow cytometer as claimed in claim 1, wherein the optics
focus the beam pattern to have a height of between about 9
micrometers and about 20 micrometers.
3. The flow cytometer as claimed in claim 1, wherein the optics
focus the beam pattern to have a height of about 20
micrometers.
4. The flow cytometer as claimed in claim 1, wherein the optics
focus the beam pattern to have a width of about 160
micrometers.
5. The flow cytometer as claimed in claim 1, wherein the detector
comprises a first detector located at 0 degrees and a second
detector located at 90 degrees relative to the path of the
irradiation beam.
6. The flow cytometer as claimed in claim 1, wherein said detector
is deferentially responsive to said light emitted from said light
emission material based upon said particle orientation
characteristics.
7. The flow cytometer as claimed in claim 1, further comprising an
analyzer coupled to said detector.
8. The flow cytometer as claimed in claim 7, wherein the analyzer
differentiates between said sperm cells based upon orientation of
said sperm cells within said fluid stream.
9. The flow cytometer as claimed in claim 7, further comprising a
droplet charger coupled to said analyzer, wherein said droplets
receive a charge differentially based upon said difference in
amount of said stain bound the nuclear DNA of X-chromosome sperm
cells and the nuclear DNA of Y-chromosome bearing sperm cells.
10. The flow cytometer as described in claim 7, further comprising
a droplet separator, wherein said droplet separator separates said
droplet based upon charge of said droplet.
11. The flow cytometer as claimed in claim 10, further comprising
at least one collection container in which droplets containing said
X-chromosome bearing sperm cells are collected as an X-chromosome
bearing population.
12. The flow cytometer as claimed in claim 10, further comprising
at least one collection container in which droplets containing
Y-chromosome bearing sperm cells are collected as a Y-chromosome
bearing population.
13. The flow cytometer as claimed in claim 1, wherein said sperm
cells are selected from a group consisting of bovine sperm cells,
equine sperm cells, and ovine sperm cells.
14. A method of sorting sperm having an associated light emission
material coupled to nuclear DNA which stains X-chromosome bearing
sperm and Y-chromosome bearing sperm in differing amounts
comprising: a) establishing a fluid stream; b) producing an
irradiation beam directed at the fluid stream; c) shaping the
irradiation beam to have a beam height between about equal to the
length of said sperm cells along the longitudinal axis and about
three times the length of said sperm cells along the longitudinal
axis at the fluid stream; and d) detecting emissions from sperm
cells passing through the beam pattern.
15. The method as claimed in claim 14, wherein the irradiation beam
is shaped to a beam pattern to having a height of between about 9
micrometers and about 20 micrometers.
16. The method as claimed in claim 14, wherein the irradiation beam
is shaped to beam pattern to having a height of about 20
micrometers.
17. The method as claimed in claim 14, wherein the irradiation beam
is shaped to beam pattern to having a width of about 160
micrometers.
18. The method as claimed in claim 14, wherein the step of
detecting emissions comprises detecting emissions at 0 degrees
position relative to the path of the irradiation beam and detecting
emissions at a 90 degrees position relative to the path of the
irradiation beam.
19. The method as claimed in claim 14, wherein the light emitted
from said light emission material varies based upon said particle
orientation characteristics.
20. The method as claimed in claim 19, further comprising the step
of analyzing the light emitted from the light emissions materials
to determine the orientation of sperm cells.
21. The method as claimed in claim 20, further comprising the step
of analyzing the light emitted from the light emissions materials
to differentiate X chromosome bearing sperm from Y chromosome
bearing sperm.
22. The method as claimed in claim 21, wherein the step of
differentiating X chromosome bearing sperm from Y chromosome
bearing sperm is performed on orientated sperm.
23. The method as claimed in claim 22, wherein one or both of the
detected X chromosome bearing sperm and Y chromosome bearing sperm
are collected in collection containers.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 13/752,057, filed on Jan. 28, 3013, which is a continuation of
U.S. application Ser. No. 12/113,684, filed on May 1, 2008, which
is a continuation of U.S. application Ser. No. 10/275,770, filed on
Feb. 7, 2003 issued as U.S. Pat. No. 7,371,517 on May 13, 2008,
which is the National Stage of International Application No.
PCT/US01/15150, filed on May 9, 2001, which claims the benefit
under 35 U.S.C. .sctn.119(e) of U.S. Provisional Application No.
60/267,571, filed Feb. 10, 2001, U.S. Provisional Application No.
60/239,752, filed Oct. 12, 2000, and U.S. Provisional Application
No. 60/203,089, filed May 9, 2000, each of which is incorporated
herein by reference.
I. TECHNICAL FIELD
[0002] Isolated high purity X-chromosome bearing or Y-chromosome
bearing populations of spermatozoa and technologies to isolate
spermatozoa, particles, or events based upon differentiation
characteristics such as mass, volume, DNA content, or the like.
II. BACKGROUND
[0003] Isolated high purity X-chromosome bearing or Y-chromosome
bearing populations of spermatozoa can be utilized to accomplish in
vitro or in vivo artificial insemination of or fertilization of ova
or oocytes of numerous mammals such as bovids, equids, ovids,
goats, swine, dogs, cats, camels, elephants, oxen, buffalo, or the
like. See also, U.S. Pat. No. 5,135,759, hereby incorporated by
reference.
[0004] However, conventional technologies for separating
spermatozoa into X-chromosome bearing and Y-chromosome bearing
populations can result in spermatozoa populations having low
purity. Regardless of the separation method spermatozoa have not
been routinely separated into X-chromosome bearing and to
Y-chromosome bearing sperm samples having high purity, such as 90%,
95%, or greater than 95%.
[0005] A number of techniques, directly or indirectly based on
differences in size, mass, or density have been disclosed with
respect to separating X-chromosome bearing from Y-chromosome
bearing spermatozoa As disclosed by U.S. Pat. No. 4,474,875, a
buoyant force is applied to all sperm cells simultaneously and
X-chromosome bearing and Y-chromosome bearing spermatozoa may then
be isolated at different locations in the separation medium. U.S.
Pat. No. 5,514,537 discloses a technique whereby spermatozoa
traverse a column packed with two sizes of beads. The larger
X-chromosome bearing spermatozoa become isolated in the layer
containing the larger beads, while the smaller Y-chromosome bearing
spermatozoa become isolated in the layer containing the smaller
beads. U.S. Pat. No. 4,605,558 discloses that spermatozoa may be
made differentially responsive to a density gradient and U.S. Pat.
No. 4,009,260 exploits the differences in migration-rate, or
swimming-speed, between the Y-bearing spermatozoa, and the
X-chromosome bearing spermatozoa, through a column of retarding
medium.
[0006] A problem common to each of the above-mentioned technologies
may be that they each act on all the spermatozoa in a
`bulk-manner`, meaning that all the spermatozoa undergo the same
treatment at the same time, and the Y-chromosome bearing sperm
cells come out faster, earlier, or at a different position than
X-chromosome bearing sperm cells. As such, individual sperm cells
may not be assessed and there may be no actual `measurement` of
volume, mass, density, or other sperm cell characteristics.
One-by-one assessment of sperm cells can provide advantages in that
the actual separation process can be monitored, and objective
quantitative data can be generated even during the separation
process, and separation parameters altered as desired. Furthermore,
these technologies may not be coupled with flow cell sorting
devices.
[0007] Flow cytometer techniques for the separation of spermatozoa
have also been disclosed. Using these techniques spermatozoa may be
stained with a fluorochrome and made to flow in a narrow stream or
band passing by an excitation or irradiation source such as a laser
beam. As stained particles or cells pass through the excitation or
irradiation source, the fluorochrome emits fluorescent light. The
fluorescent light may be collected by an optical lens assembly,
focused on a detector, such as a photomultiplier tube which
generates and multiplies an electronic signal, which may then be
analyzed by an analyzer. The data can then be displayed as multiple
or single parameter chromatograms or histograms. The number of
cells and fluorescence per cell may be used as coordinates. See
U.S. Pat. No. 5,135,759, hereby incorporated by reference. However,
with respect to this type of technology a variety of problems
remain unresolved and isolating highly purified populations of
X-chromosome bearing or Y-chromosome bearing sperm cells remains
difficult.
[0008] A significant problem with conventional flow cytometer
technologies can be the orientation of objects, particles, or cells
in the sheath fluid stream. This can be particularly problematic
when the object or cell is irregular in shape with respect to more
than one axis, such spermatozoa for example. One aspect of this
problem may be establishing the initial orientation of the object
within the sheath fluid stream. A second aspect of this problem may
be maintaining the orientation of the object with respect to the
detector (photomultiplier tube or otherwise) during the period that
emitted light from the object is measured.
[0009] Another significant problem with conventional flow cytometer
technologies can be the failure to encapsulate the objects or cells
in a droplet of liquid. Especially, when droplets are formed around
irregularly shaped objects the droplet may not be of sufficient
size to completely surround all the features of the objects or
cells. For example, during flow cytometry operation as
above-described droplets can be formed at very high speed, even as
many as 10,000 to 90,000 droplets per second and in some
applications as many as 80,000 droplets per second. When
spermatozoa are encapsulated into droplets, especially at these
high rates of speed, a portion of the tail or neck may not be
encapsulated in the droplet. That portion of the tail or neck not
encapsulated in the droplet may then be responsive with the nozzle
or may be responsive to the environment surrounding the droplet in
a manner that interferes with subsequent droplet formation or with
proper deflection of the droplet. As a result some of the
spermatozoa may not be analyzed at all reducing the efficiency of
the procedure, or may not be resolved sufficiently to be assigned
to a population, or may be deflected in errant trajectories, or a
combination of all may occur.
[0010] Another significant problem with conventional flow cytometer
technologies, as well as other technologies, can be a coincidence
of measurable events. One aspect of this problem can be that the
incident light flux from a first event continues to produce signals
after the incident light flux from a second event starts to
generate a signal. As such, the two events remain at least
partially unresolved from one another. Another aspect of this
problem can be that two or more events are simultaneously initiated
and the incident light flux comprises the contribution of all the
events. As such, the multiplicity of events may not be resolved at
all and the objects corresponding to the multiplicity of events can
be incorrectly assigned to a population or not assigned to a
population at all, or both. Specifically, with respect to flow
cytometry, individual particles, objects, cells, or spermatozoa in
suspension flow through a beam of light with which they interact
providing a measurable response, such as fluorescent emission. In
conventional flow cytometry, Hoechst stained spermatozoa traverse a
laser beam resulting in a fluorescent light emission. The
fluorescent light emission from the excited fluorochrome bound to
the DNA can be bright enough to produce an electron flow in
conventional photomultiplier tubes for a period of time after the
actual emission event has ended. Moreover, in a conventional flow
cytometer, the laser beam can produce a pattern having a height of
30 .mu.m while the width can be approximately 80 .mu.m. The nucleus
of a bovine spermatozoa which contains fluorochrome bound DNA can
be about 9 .mu.m in length making the height of the laser beam some
three (3) times greater than the nucleus. This difference can allow
for the laser excitation of the bound fluorochrome in more than one
spermatozoa within the laser beam pattern at one time. Each of
these conventional flow cytometry problems decreases the ability to
resolve individual events from one another.
[0011] Another significant problem with conventional flow cytometer
technologies, and other technologies, can be that irregularly
shaped objects, such as spermatozoa, generate differing signals
(shape, duration, or amount) depending on their orientation within
the excitation/detection path. As such, individuals within a
homogenous population can generate a broad spectrum of emission
characteristics that may overlap with the emission characteristics
of individuals from another homogenous population obviating or
reducing the ability to resolve the individuals of the two
populations.
[0012] Another significant problem with conventional flow cytometer
technologies, and other technologies, can be that objects are not
uniformly exposed to the excitation source. Conventional beam
shaping optics may not provide uniform exposure to laser light when
the objects are close to the periphery of the beam.
[0013] Another significant problem with conventional flow cytometer
technologies can be that objects, such as spermatozoa, can be
exposed to the excitation source for unnecessarily long periods of
time. Irradiation of cells, such as spermatozoa, with laser light
may result in damage to the cells or to the DNA contained within
them.
[0014] Another significant problem with conventional flow cytometer
technologies can be that there may be a disruption of the laminar
flow within the nozzle by the injection tube. Disruption of the
laminar flow can change the orientation of irregularly shaped
objects within the flow and lower the speed of sorting and the
purity of the sorted populations of X-chromosome bearing sperm or
Y-chromosome bearing spermatozoa.
[0015] There may be additional problems with technologies that
utilize stain bound to the nuclear DNA of sperm cells. First,
because the DNA in the nucleus is highly condensed and flat in
shape, stoichiometric staining of the DNA may be difficult or
impossible. Second, stained nuclei may have a high index of
refraction. Third, stain bound to the DNA to form a DNA-stain
complex may reduce fertilization rates or the viability of the
subsequent embryos. Fourth, the DNA-stain complex is typically
irradiated with ultra-violet light to cause the stain to fluoresce.
This irradiation may affect the viability of the spermatozoa. Due
to these various problems, it may be preferable to use a method
that requires less or no stain, or less or no ultra-violet
radiation, or less or none of both.
[0016] With respect to generating high purity samples of
X-chromosome bearing sperm cell or Y-chromosome bearing sperm cell
populations (whether live, fixed, viable, non-viable, intact,
tailless, or as nuclei), or generally, with respect to detecting
small differences in photo-generated signal between serial events
having relatively high incident light flux, or with respect to
orienting irregularly shaped objects in a fluid stream, or
eliminating coincident events within an optical path, or removing
undesirably oriented objects from analysis, the instant invention
addresses every one of the above-mentioned problems in a practical
fashion.
III. DISCLOSURE OF THE INVENTION
[0017] A broad object of the invention can be to provide isolated
high purity X-chromosome bearing and Y-chromosome bearing
populations of spermatozoa. Isolated non-naturally occurring
populations of spermatozoa that have high purity have numerous
applications including sex selection of offspring from mammals,
various in vitro protocols for the fertilization of ova, various in
vivo protocols such as artificial insemination, business methods
involving the production of prize animals or meat animals, or
preservation of rare or endangered animals, to recite but a few of
the applications for high purity populations of spermatozoa.
[0018] Another broad object of the invention involves both devices
and methods for the production of high purity X-chromosome bearing
and Y-chromosome bearing sperm samples.
[0019] Particular embodiments of the invention are described, which
may be used in numerous applications as above-mentioned, that can
be used to achieve the specific objects of differentiating between
bright photoemissive events having small measurable differences in
total light flux, orienting irregularly shaped objects in a fluid
stream, the minimization of coincident events within an optical
path, the removal of signal contributed by undesired unoriented
objects within an optical path (including the removal of the object
itself), and the encapsulation of irregularly shaped objects within
a droplet. As such, the specific objects of the invention can be
quite varied.
[0020] Another broad object of the invention can be to provide
X-chromosome bearing or Y-chromosome bearing spermatozoa samples
(live, fixed, viable, non-viable, intact, tailless, or sperm
nuclei) having a graded level of high purity in the range of 80%,
85%, 90%, 95%, or even greater than 95%.
[0021] Another significant object of particular embodiments of the
invention can be to sort spermatozoa into X-chromosome bearing and
Y-chromosome bearing populations having high purity even at high
separation rates. The high speed separation can produce live sperm
of each sex at rates of about 500, 1000, 2000, 3000, 4000, 5000,
6000, 7000, 8,000, 9,000 or even 10,000 per second, or higher.
[0022] Another significant object of particular embodiments of the
invention can be to substantially eliminate or remove spermatozoa
(live, fixed, viable, non-viable, intact, tailless, or sperm
nuclei) having undesired orientation in the excitation/detection
portion of the flow path of a flow cytometer.
[0023] Another significant object of particular embodiments of the
invention can be to provide artificial insemination samples of
X-chromosome bearing or Y-chromosome bearing spermatozoa having a
high level of purity.
[0024] Another significant object of particular embodiments of the
invention can be to provide in vitro insemination samples of
X-chromosome bearing or Y-chromosome bearing spermatozoa having a
high level of purity.
[0025] Another significant object of a particular embodiment of the
invention can be to preselect the sex of offspring of females
inseminated with high purity artificial insemination samples, the
sex of offspring of ova fertilized with high purity artificial
insemination samples, with selection success rates of 80%, 85%,
90%, 95%, or greater than 95%.
[0026] Another significant object of particular embodiments of the
invention can be to differentiate between photoemissive events
having small differences in total emitted light flux.
[0027] Another significant object of particular embodiments of the
invention can be to substantially eliminate or reduce the amount of
background noise generated by a photomultiplier tube, even in the
absence of light, during the period after exposure to high incident
light flux.
[0028] Another significant object of particular embodiments of the
invention can be to substantially eliminate saturation of the
photocathode of photomultiplier tube(s) used in conjunction with
flow cytometry, or otherwise.
[0029] Another significant object of particular embodiments of the
invention can be to reduce the number electrons migrating from the
photocathode of a photomultiplier tube to the first dynode.
[0030] Another significant object of particular embodiments of the
invention can be to reduce the total flow of electrons to the N
electrode of a photomultiplier tube.
[0031] Another significant object of particular embodiments of the
invention can be to allow increased light flux to the photocathode
of the photomultiplier tube without proportionately increasing the
amount of background signal generated by the photomultiplier
tube.
[0032] Another significant object of particular embodiments of the
invention can be to increase the signal to background signal ratio
from measured photoemissive events.
[0033] Another significant object of particular embodiments of the
invention can be to allow increased amplification of the signal
generated from the photomultiplier tube during high incident light
flux events or serial high incident light flux events without
saturating the photocathode of the photomultiplier tube.
[0034] Another significant object of particular embodiments of the
invention can be to increase the apparent resolution of
chromatograms or histograms resulting from sorting fluorochrome
stained sperm, or other cells, or other objects, having small
differences in emitted light flux upon excitation of the bound
fluorochrome(s).
[0035] Another significant object of particular embodiments of the
invention can be to improve the calibration of sorting flow
cytometer instruments when used for sorting spermatozoa.
[0036] Another significant object of particular embodiments of the
invention can be to increase the sperm sorting rate of flow
cytometer systems.
[0037] Another significant object of particular embodiments of the
invention can be to increase the purity of the sperm samples sorted
by flow cytometry.
[0038] Another significant object of particular embodiments of the
invention can be to provide techniques for the sorting of
X-chromosome bearing sperm from Y-chromosome bearing sperm where
there is a small difference in the amount of Y chromosome DNA to
the amount of X chromosome DNA relative to the total amount of
nuclear DNA.
[0039] Another significant object of particular embodiments of the
invention can be to provide techniques which improve the apparent
resolution of histograms generated during the process of sorting
X-chromosome bearing sperm from Y-chromosome bearing sperm with a
flow cytometer.
[0040] Another significant object of particular embodiments of the
invention can be to provide beam shaping optics which minimizes
coincidence of objects within the excitation detection path.
[0041] Another significant object of particular embodiments of the
invention can be to provide beam shaping optics that minimizes the
total lumens an object is exposed to traversing the excitation
beam. One aspect of this object can be to decrease the total lumens
an object is exposed to. A second aspect of this object can be to
increase the power of the light source without increasing the total
lumens the object is exposed to.
[0042] Another significant object of particular embodiments of the
invention can be to provide beam shaping optics that allow for
uniform exposure of objects that pass through the optical path.
[0043] Another significant object of particular embodiments of the
invention can be to provide a nozzle that orients irregularly
shaped objects in a fluid stream. One aspect this object can be to
orient elongated objects in the same direction. A second aspect of
this object can be to orient dorso-laterally flatted objects in the
same direction.
[0044] Another significant object of particular embodiments of the
invention can be to fully encapsulate irregularly shaped objects
within a drop of fluid.
[0045] Another significant object of particular embodiments of the
invention can be to differentiate undesirably oriented objects from
desirably oriented objects in a fluid stream.
[0046] Another object of an embodiment of the invention can be to
provide differential interference contrast technology, whereby the
object-plane consists of a fluid stream carrying the objects of
interest, and whereby the image-plane can be used to measure the
signal from the passing objects.
[0047] Another object of an embodiment of the invention can be to
provide optics that form two laterally separated images from each
object in such a way that one can be used to measure the actual
volume, and one to determine the orientation. This way, objects
that were not orientated properly to allow an accurate measurement
of its volume can be discarded. This can be accomplished by
modifications so that the light pulses, resulting from these two
images can be detected independently using two pinholes in the
image plane. Optics are tuned in such a way that a first image can
give rise to a light pulse proportional to the volume of the
object, and that a second image can give rise to a light pulse
dependent on the orientation the object had when it was
measured.
[0048] Another object of an embodiment of the invention can provide
a manner of compensating for the fact that the objects are
contained inside a fluid stream. The fluid stream can be a cylinder
of water, for example, which acts as a cylindrical lens, thus
distorting the image of the object. Optically, this corresponds to
cylinder of higher refractive index (water) than its surroundings
(air). The compensation disclosed in this invention can consist of,
for example, a cylinder having a refractive index lower than its
surroundings, although other compensating elements of various
shapes and refractive index may also be designed as the need
requires. By making sure the light passes through this compensation
element, the optical effect of the fluid stream can be compensated
by the exactly opposite behavior of the compensation element.
[0049] Naturally further objects of the invention are disclosed
throughout other areas of the specification and claims.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 shows a generalized flow cytometer.
[0051] FIG. 2 shows a second view of a generalized flow
cytometer.
[0052] FIG. 3 shows a comparison of univariate histograms from flow
cytometers (#1, #2, and #3) without the amplifier invention (FIG.
3A) with univariate histograms for the same flow cytometers using a
particular embodiment of the amplification invention (FIG. 3B)
illustrating the improved resolution between X-chromosome bearing
and Y-chromosome bearing populations of bovine spermatozoa.
[0053] FIG. 4 shows univariate and bivariate histograms
illustrating the conventional resolution between X-chromosome
bearing and Y-chromosome bearing populations of bovine
spermatozoa.
[0054] FIG. 5 shows univariate and bivariate histograms
illustrating improved resolution between X-chromosome bearing and
Y-chromosome bearing populations of bovine spermatozoa using a
particular embodiment of the amplification invention.
[0055] FIG. 6 shows a second example of univariate and bivariate
histograms illustrating the conventional resolution between
X-chromosome bearing and Y-chromosome bearing populations of bovine
spermatozoa.
[0056] FIG. 7 shows a second example of univariate and bivariate
histograms illustrating the improved resolution between
X-chromosome bearing and Y-chromosome bearing populations of bovine
spermatozoa using a particular embodiment of the amplification
invention.
[0057] FIG. 8 shows univariate and bivariate histograms
illustrating the conventional resolution between X-chromosome
bearing and Y-chromosome bearing populations of equine
spermatozoa.
[0058] FIG. 9 shows univariate and bivariate histograms
illustrating the improved resolution between X-chromosome bearing
and Y-chromosome bearing populations of equine spermatozoa using a
particular embodiment of the amplification invention.
[0059] FIG. 10 shows univariate and bivariate histograms
illustrating the improved resolution between X-chromosome bearing
and Y-chromosome bearing populations of equine spermatozoa nuclei
using a particular embodiment of the amplification invention.
[0060] FIG. 11 shows a particular embodiment of the circuit board
modification to make the amplification invention with respect to a
MoFlo.RTM. flow cytometer.
[0061] FIG. 12 shows an electrical schematic diagram of a
particular embodiment of the amplification invention with respect
to a MoFlo.RTM. flow cytometer.
[0062] FIG. 13 shows the laser beam pattern using conventional beam
shape optics (FIG. 13A) and the laser beam pattern using the
reduced height beam shape optics (FIG. 13B).
[0063] FIG. 14 shows a bar graph that compares the purity of
separated X-chromosome bearing spermatozoa (FIG. 14A) and
Y-chromosome bearing spermatozoa (FIG. 14B) using conventional
technology or using the amplification invention independently or in
conjunction with reduced height beam shaping optics.
[0064] FIG. 15 shows a front view of the reduced height beam
shaping optics.
[0065] FIG. 16 shows a top view of the reduced height beam shaping
optics.
[0066] FIG. 17 shows a perspective (FIG. 17A) and one cross-section
(FIG. 17B) of the object orienting nozzle invention.
[0067] FIG. 18 shows a graded series of cross sections (CC-NN) of
the object orienting nozzle invention.
[0068] FIG. 19 shows a front view and an end view of an embodiment
of the beveled injection tube invention.
[0069] FIG. 20 illustrates the removal of undesired unoriented
spermatozoa (RUUS) invention by comparison of signal(s) from the
oriented spermatozoa (FIGS. 20A and 20B) and the signal(s) from the
unoriented spermatozoa (FIGS. 20C and 20D).
[0070] FIG. 21A shows a perspective of another embodiment of the
beveled injection tube invention having a paddle shaped beveled
blade and FIG. 21B shows a cross-section of the injection tube.
[0071] FIG. 22 shows a conventional optics technology coupled to a
flow cytometer.
[0072] FIG. 23A shows the shape and size of a typical spermatozoon
and FIG. 23B shows the difference between correctly and
non-correctly orientated spermatozoa.
[0073] FIG. 24 shows an embodiment of the invention having
construction allowing the measurement of two signals, for example
volume and orientation.
[0074] FIGS. 25A and B shows an embodiment of the invention having
two halves with a pinhole corresponding to each half, FIG. 25C
shows an image plane of an embodiment of the invention, FIG. 25D
shows an embodiment of the invention having two independently
rotatable polarizers.
[0075] FIGS. 26A and 26B illustrates the compensation method for
the fluid stream for an embodiment of the invention, FIG. 26C shows
an embodiment of a compensation element, 26 D shows another
embodiment of a compensation element where images of a fluid stream
and from the compensation element fall on top of each other in the
image plane.
[0076] FIG. 27 shows an embodiment of the interference optics
invention.
[0077] FIG. 28 shows a second view of the interference optics
invention.
V. MODE(S) FOR CARRYING OUT THE INVENTION
[0078] The invention involves isolated high purity X-chromosome
bearing and Y-chromosome bearing populations of spermatozoa or
sperm cells. High purity X-chromosome bearing and Y-chromosome
bearing populations of spermatozoa can comprise populations of
intact live spermatozoa, and may also comprise populations of
tailless spermatozoa (sperm nuclei), or populations of other viable
or non-viable forms of spermatozoa, as may be desired. While
particular examples are provided that describe the invention in the
context of separating intact live sperm cells each having a sperm
cell head, necks, and tail, it should be understood that the
technologies described can have various applications with respect
to sperm nuclei as well. X-chromosome bearing and Y-chromosome
bearing populations of spermatozoa should further be understood to
encompass spermatozoa from any male of a species of mammal
including, but not limited to, spermatozoa from humans and
spermatozoa from commonly known animals such as bovids, equids,
ovids, canids, felids, goats, or swine, as well as less commonly
known animals such as elephants, zebra, camels, or kudu. This list
of animals is intended to be exemplary of the great variety of
animals from which spermatozoa can be routinely sorted at 90% or
greater purity, and is not intended to limit the description of the
invention to the spermatozoa from any particular species of
mammals.
[0079] High purity separated spermatozoa from the various species
of mammals can be incorporated into products that can be used with
artificial insemination protocols or as part of commercial business
methods such as those as described in U.S. Patent Application Nos.
60/211,093, 60/224,050, or Patent Cooperation Treaty Application
No. US99/17165; or be used with low dose insemination protocols as
described in Patent Cooperation Treaty Application No. US98/27909,
or used in vitro fertilization of oocytes from animals, including
humans, as described in U.S. Patent Application No. 60/253,785,
each of the above-mentioned references are hereby incorporated by
reference.
[0080] The use of the term purity or high purity should be
understood to be the percent of the isolated spermatozoa population
bearing a particular differentiating characteristic or desired
combination of characteristics. For example, where a population of
spermatozoa are separated based upon bearing an X-chromosome as
opposed to a Y-chromosome, an X-chromosome bearing population
having 90% purity comprises a population of spermatozoa of which
90% of the individual spermatozoa bear an X-chromosome while 10% of
such population of spermatozoa may bear a Y-chromosome. As such,
high purity with respect to X-chromosome bearing populations or
Y-chromosome bearing populations can comprise a purity selected
from the group consisting of between 90% to about 100%, between
about 91% to about 100%, between about 92% to about 100%, between
about 93% to about 100%, between about 94% to about 100%, between
about 95% to about 100%, between about 96% to about 100%, between
about 97% to about 100%, between about 98% to about 100%, between
about 99% to about 100%.
[0081] Importantly, while numerous embodiments of the invention
describe isolated high purity X-chromosome and Y-chromosome bearing
populations of spermatozoa, and while the description further
discloses high purity spermatozoa separation devices and methods of
how to isolate and how to use isolated high purity populations of
spermatozoa, the basic concepts of the invention should be
understood to be applicable to other types of particles or events
having particle differentiation characteristics or event
differentiation characteristics. It should be understood that the
invention can be applicable to a variety of circumstances in which
resolving small differences in photogenerated signal may be
necessary, such as product defect detection, field flow
fractionation, liquid chromatography, electrophoresis, computer
tomography, gamma cameras, time of flight instruments, or the like
as would be readily understood by those skilled in those arts.
[0082] Moreover, while this disclosure provides descriptions of
embodiments of apparatus and methods for flow separation of
X-chromosome bearing spermatozoa from Y-chromosome bearing
spermatozoa, the description of these embodiments of the invention
is not meant to reduce the scope of the invention to only flow
separation of spermatozoa or only to high purity flow cytometer
spermatozoa separation systems but rather these examples are
intended to exemplify the basic concepts of the invention in a
practical manner so that they may be applied to the wide variety of
applications.
[0083] Now referring to FIGS. 1 and 2, a flow cytometer embodiment
of the invention is shown which includes a particle or cell source
(1) which acts to establish or supply particles or cells stained
with at least one fluorochrome for analysis. The particles or cells
are deposited within a nozzle (2) in a manner such that the
particles or cells are introduced into a fluid stream or sheath
fluid (3). The sheath fluid (3) is usually supplied by some sheath
fluid source (4) so that as the particle or cell source (1)
supplies the particles or cells into the sheath fluid (4) they are
concurrently fed through the nozzle (2).
[0084] In this manner it can be easily understood how the sheath
fluid (3) forms a sheath fluid environment for the particles or
cells. Since the various fluids are provided to the flow cytometer
at some pressure, they flow out of nozzle (2) and exit at the
nozzle orifice (5). By providing some type of oscillator (6) which
may be very precisely controlled through an oscillator control (7),
pressure waves may be established within the nozzle (2) and
transmitted to the fluids exiting the nozzle (2) at nozzle orifice
(5). Since the oscillator (6) acts upon the sheath fluid (3), the
stream (8) exiting the nozzle orifice (5) 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, and
can be sperm cells with respect to some embodiments of the
invention.
[0085] Since the drops (9) 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 sensing system (10). The
particle or cell sensing system involves at least some type of
detector or sensor (11) which responds to the particles or cells
contained within fluid stream (8). The particle or cell sensing
system (10) 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 laser
exciter (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 dependent 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.
[0086] In order to achieve separation and isolation based upon
particle or cell characteristics, emitted light can be received by
sensor (11) and fed to some type of separation discrimination
system or analyzer (13) coupled to a droplet charger which
differentially charges each droplet (9) based upon the
characteristics of the particle or cell contained within that
droplet (9). In this manner the separation discrimination system or
analyzer (13) acts to permit the electrostatic deflection plates
(14) to deflect drops (9) based on whether or not they contain the
appropriate particle or cell.
[0087] As a result, the flow cytometer acts to separate the
particle or cells (16) by causing them to be directed to one or
more collection containers (15). 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. patent application Ser. No. 09/001,394, hereby
incorporated by reference herein. Naturally, numerous deflection
trajectories can be established and collected simultaneously.
[0088] To routinely separate particles, cells, sperm cells, or
spermatozoa (intact, live, fixed, viable, non-viable, or nuclei)
into high purity X-chromosome bearing and Y-chromosome bearing
populations, the particle differentiation apparatus or methods used
must provide high resolution of the differentiation characteristics
that are used as the basis of analysis and separation.
[0089] With respect to spermatozoa, differentiating between the
light emitted by the fluorochrome bound to the nuclear DNA of
X-chromosome bearing sperm cells and the light emitted by the
fluorochrome bound to the nuclear DNA of Y-chromosome bearing sperm
cells may be difficult as discussed above.
[0090] In many applications, the total emitted light from
photoemissive events incident to the detector, which can be a
photomultiplier tube, can be high while the difference between the
emitted light of each photoemissive events to be differentiated can
be small. The problem can be exacerbated when the photoemissive
events happen serially at high rate of speed and the time period
between photoemissive events is short, such as with high speed cell
sorting using flow cytometers. When separating particles, cells, or
sperm cells based upon the difference in bound fluorochrome the
cells flow past an excitation source and a high number of emissive
events per second can be established. As a result, the amount of
emitted light generated in the stream of particles, cells, or sperm
cells, can be enormous. As the speed of the stream is increased,
the intercept point with the excitation source becomes very bright.
This high level of incident light upon the photocathode of the
photomultiplier tube can cause a very low signal to background
signal ratio. The amount of background signal can be further
exacerbated when fluorochrome such as Hoechst 33342 can be used to
label the nuclear DNA of sperm cells.
[0091] Most solutions to the problem have focused on decreasing the
total amount of light flux upon the photocathode tube by placing
optical filters in front of the photomultiplier tube. This approach
does not change the proportion of signal to background signal and
subsequent attempts to increase the sensitivity of the
photomultiplier tube generates additional background signal as the
photomultiplier tube saturates from the amount of background
signal.
[0092] Typically, photomultiplier tubes have an operation voltage
range of about 400 volts to about 900 volts. The lower limit of
linear operation of standard photomultiplier tubes, such as the
R928 and R1477 photomultiplier tubes available from Hamamatsu
Corporation, may be about 300 volts. As such, equipment or
instruments which employ photomultiplier tubes are configured to
operate such photomultiplier tubes at or above 400 volts. Even
where reduction of the number of electrons at the anode is desired,
as disclosed in U.S. Pat. Nos. 4,501,366 and 5,880,457 the voltage
between the photocathode and the first dynode is maintained at a
high voltage and reduction of the electrons at the anode is
accomplished by either decreasing the voltage to the remaining
dynodes, or the inherent dark noise or shot noise is filtered out
electronically.
[0093] Unexpectedly, reducing the amount of voltage to the
photomultiplier tube below 400 volts to about 280 volts, or about
250 volts, or even to just above 0 volts can allow small
differences in photoemissive light to be differentiated even when
the total light emitted from each photoemissive event is high, or
even when there are a high number of bright serial events per
second. With respect to the rate of photoemmissive events generated
from the irradiation of fluorochromes bound to the nuclear DNA of
spermatozoa, the invention allows the rate of photoemmissive events
that can be achieved during separation of spermatozoa into
X-chromosome bearing and Y-chromosome bearing populations to be
increased to a separable event rate of at least 5000 separable
events per second, at least 6000 separable events per second, at
least 7000 separable events per second, at least 8000 separable
events per second, at least 9000 separable events per second, at
least 10,000 separable events per second, at least 11,000 separable
events per second, at least 12,000 separable events per second, at
least 13,000 separable events per second, at least 14,000 separable
events per second, at least 15,000 separable events per second, at
least 16,000 separable events per second, at least 17,000 separable
events per second, at least 18,000 separable events per second, at
least 19,000 separable events per second, at least 20,000 separable
events per second, at least 25,000 separable events per second, at
least 30,000 separable events per second, and at least 35,000
separable events per second, or greater.
[0094] As a specific example, existing Cytomation SX MoFlo.RTM.
sorting flow cytometers are configured to operate the
photomultiplier tube at 400 volts minimum. The gain can be adjusted
to operate the photomultiplier tube at higher voltages but not
lower voltages. SX MoFlo.RTM. flow cytometers can be converted by
reconfiguring the photomultiplier controllers. The R16C resistor
(2.49 kiloohms) on channel three can be replaced by a 2.0 k.OMEGA.
resistor to alter the gain of the amplifier that controls the
photomultiplier tube. This conversion allowed the photomultiplier
tube to be operated at about 280 volts. Similar conversion of SX
MoFlo.RTM. flow cytometers with two 3.75 k.OMEGA. resistors in
parallel, or a 1.3 k.OMEGA. resistors can allow the photomultiplier
tube to be operated at voltages of about 200 volts, or just above
zero volts, respectively. Also with respect to this conversion, the
neutral density filter in front of the photocathode can also be
removed as a result of operating the photomultiplier tube outside
of the typical operation voltage range.
[0095] This conversion unexpectedly increases the signal to noise
ratio of the photoemissive event as it is translated to an
electronic signal by the photomultiplier tube. The cleaner signal
may then be amplified by increasing the gain amplifier to the
analog to digital converter of the analyzer (13) to the appropriate
level and output may be generated as univariate or bivariate
histograms.
[0096] Now referring to FIG. 3, a comparison of univariate
histograms generated on three different SX MoFlo.RTM. flow
cytometers (#1, #2, #3) prior to the use of the invention (FIG.
1A), and using the invention (FIG. 1B) with respect to the
separation of intact live ejaculated bovine sperm are shown. As can
be understood from the univariate histograms, the resolution (the
apparent differentiation of the X-chromosome bearing population
from the Y-chromosome bearing population represented by the valley
between peaks) of intact live X-chromosome bearing spermatozoa (17)
from live Y-chromosome bearing spermatozoa (18) can be
substantially improved by use of the invention.
[0097] The mean separation rate or sort rates of intact live
spermatozoa prior to use of this embodiment of the invention with
the SX MoFlo.RTM. flow cytometers was about 17.9.times.10.sup.6/4.5
hours of both X-chromosome bearing spermatozoa and Y-chromosome
bearing spermatozoa (i.e. about 1,100 separations or sorts per
second in each of two streams--the first stream X-chromosome
bearing spermatozoa and the second stream Y-chromosome bearing
spermatozoa) at about 87% purity with a range of 84% to 93% purity.
The separable event rate was 22,000, 23,000, and 20,000
respectively for the three sorts.
[0098] The mean sort rates of live spermatozoa after the
above-mentioned conversion was about 40.3.times.106/4.5 hour sort
(i.e. about 2,500 sorts per second per stream) at about 90.8%
purity with a range of 89% to about 92%. The events per second were
13,000, 15,000, and 19,500 respectively for the three sorts.
[0099] As can be understood from the data not only did this
embodiment of the invention result in increased purity of the
separated spermatozoa populations but also allowed the separation
rate or sort rate to be more than doubled while the separable
events rate was actually decreased.
[0100] Similarly, referring now to FIGS. 4 and 5, which show
bivariate histograms from sorting of intact live bull spermatozoa
with the SX MoFlo.RTM. flow cytometer #1 prior to using the
invention (FIG. 4) and after the above-mentioned conversion (FIG.
5). Prior to using the invention, the SX MoFlo.RTM. flow cytometer
was initially operated at 440 volts at the photocathode with the
laser adjusted to 135 MW, a gain of 1.times. and with a neutral
density filter of 1.0 ( 1/10th amplitude) at about 10,000 events
per second. Upon using the invention, the SX MoFlo.RTM. flow
cytometer was operated at about 262 volts at the photocathode, with
the laser adjusted to about 100 mW, a gain of 4.times., without the
neutral density filter, at about 10,000 separable events per
second. As can be understood from this data there is a large
increase in resolution as evidenced by the increased depth of the
valley between the X-chromosome bearing population (19) and the
Y-chromosome bearing population (20).
[0101] Similarly, referring now to FIGS. 6 and 7, which show
bivariate histograms from sorting of intact live bull spermatozoa
with the SX MoFlo.RTM. flow cytometer #2 before using this
embodiment of the invention (FIG. 6) and upon using this embodiment
of the invention (FIG. 7) operated at the same parameters as shown
in FIGS. 3 and 4 respectively. Again, there can be a large increase
in resolution as evidenced by the depth of the valley between the
X-chromosome bearing population (21) and the Y-chromosome bearing
population (22).
[0102] Now referring to FIGS. 8 and 9, which show bivariate
histograms from separation or sorting of intact live equine
spermatozoa with the SX MoFlo.RTM. flow cytometer before using this
embodiment of the invention (FIG. 8) and upon using this embodiment
of the invention (FIG. 9). When using this embodiment of the
invention, live equine spermatozoa were separated or sorted with
the laser power at 100 mW with the photomultiplier tube voltage
below 300 volts. The separation rates or sort rates exceeded 4,800
sorts per second average at 12,000 events per second. The increased
resolution of the X-chromosome bearing population (23) and the
Y-chromosome bearing population (24) is dramatic. The data shows
that about 8 to about 9 channels separation can be achieved with
this embodiment of the invention as compared to 5 channels of
separation between the peaks without the use of this embodiment of
the invention. The purity of both the sorted X-chromosome bearing
population and the sorted Y-chromosome bearing population was about
93%.
[0103] Now referring to FIG. 10, which shows a univariate histogram
and a bivariate dot plot from sorting of Hoechst 33342 stained
stallion sperm nuclei (S-05400) separated using this embodiment of
the invention. The nuclei were prepared from freshly ejaculated
stallion sperm. The sperm were washed by centrifugation, sonicated
and the resultant heads and tails separated using Percoll density
gradient centrifugation. The isolated heads were washed, fixed with
2% formalin and then stained with Hoechst 33342. The stained nuclei
were stabilized using sodium azide (0.5%). The sample was run at
5000 events per second to produce the histograms. The stained
nuclei were then used to calibrate an SX MoFlo.RTM. flow cytometer
was converted as above-mentioned to incorporate the photomultiplier
tube embodiment of the invention. Compensation was used to level
the two populations (X stained nuclei and Y stained nuclei) in the
bivariate plot. Note that the two populations of equine sperm
nuclei are nearly fully resolved to baseline as shown by the
univariate plot.
[0104] Now referring to FIG. 11, a modification specifically for SX
MoFlo.RTM. flow cytometer includes the use of two resistors in
parallel to provide the correct value of 1.8 k.OMEGA.. Two 3.57
k.OMEGA. resistors (25) and (26) are equal to about 1.785 k.OMEGA.
which can be sufficiently close to the value to be effective. With
this modification the photomultiplier tube on this particular
instrument can then be run at about 200 volts. Naturally, a similar
modification can be made to other flow cytometer instruments or
other instruments which use a photomultiplier tube to measure the
amount of light emitted from particular events. FIG. 12, provides
an electrical schematic diagram for this particular embodiment of
the invention.
[0105] Another important embodiment of the invention can be a
reduced height irradiation beam pattern optics. As shown by FIG.
13A, conventional irradiation beam shaping optics generate a beam
pattern (27) that can have a height can be greater than much
greater than the height of the sperm cell head(s) (28) passing
through it. As a result, more than a single sperm cell head
containing fluorochrome bound DNA can enter the irradiation beam
pattern at the same time. In that case, the fluorochrome(s) bound
to the DNAs contained within the multiple sperm heads can be
excited simultaneously and fluoresce within a single emissive
event. As such, the prior or subsequent emissive event can include
coincident light flux contributed from other sperm head(s) in the
beam pattern (27). This results in a reduced difference in mean
light flux between light emissive events which distinguish between
X-chromosome bearing spermatozoa and Y-chromosome bearing
spermatozoa. It can also decrease the difference in mean light flux
between events that compare light emissions of X-chromosome bearing
spermatozoa or Y-chromosome bearing spermatozoa. Importantly,
coincident excitation of fluorochrome bound to multiple DNAs
increases the mean brightness of the events making the measurable
difference in light flux between events an even smaller percentage
of the total light flux emitted. This makes quantification of the
differences between events even more difficult.
[0106] By reducing the height of the beam shape as shown by FIG.
13B, the coincidence of multiple sperm heads being within the
reduced height beam (29) pattern during the same measured event is
reduced. This results in an increased mean difference between light
emissive events which distinguish between X-chromosome bearing
spermatozoa and Y-chromosome bearing spermatozoa. It can also
reduce the mean total light flux for each measured emissive event.
For particular embodiments of the invention used for sorting bovine
sperm which have a nucleus of about 9 .mu.m, it has been found that
the height of the beam can be about 20 .mu.m. In this application,
it has been found that vertical beam heights of less than 20 .mu.m
did not provide an additional gain in resolution.
[0107] Referring to FIG. 14, it can be understood that the use of
reduced height irradiation beam pattern optics can improve the
purity of sorted populations of X-chromosome bearing bovine
spermatozoa (FIG. 14A) and sorted populations of Y-chromosome
bearing bovine sperm (FIG. 14B) that have been stained with Hoechst
33342 stain. This is true for both 25% and 40% sort gates of the
univariate peak. As can be further understood from FIG. 14, the
reduced height beam pattern optics can improve purity of separated
spermatozoa independent of any other aspect of the invention, such
as modification of photomultiplier circuitry embodiment of the
invention (new PMT) as described above, or can be used in
conjunction with the modified photomultiplier embodiment of the
invention to increase the purity of separated spermatozoa samples
even further.
[0108] Another advantage of the reduced height beam pattern optics
can be that the transit time of the spermatozoa in the excitation
laser beam or irradiation beam can be reduced. A reduced amount of
irradiation time within the excitation laser beam may result in
less stress or damage to the spermatozoa.
[0109] Again referring to FIG. 14B, it can be understood that the
reduced height beam pattern can be used in conjunction with an
irradiation beam pattern having greater area than conventionally
used. For example, conventional beam patterns (27), such as that
shown in FIG. 14A, have an elliptical pattern of about 30
.mu.m.times.80 .mu.m while the invention when used for sorting
bovine sperm generates optimal resolution between X-chromosome
bearing and Y-chromosome bearing populations when the beam has a 20
.mu.m.times.160 .mu.m beam pattern (29). The 20 .mu.m.times.160
.mu.m beam pattern has approximately 1.3 times the area of the 30
.mu.m.times.80 .mu.m beam pattern. As such, there can be an inverse
proportion in loss of energy at the incident point. This makes it
possible to increase the excitation laser power without concern for
increasing the irradiation damage to the spermatozoa. For example,
if an instrument has conventional beam shaping optics that produce
a 30 .mu.m.times.80 .mu.m irradiation beam pattern and the
excitation laser is conventionally powered at 150 mW, then
particular embodiments of the invention with a 20 .mu.m.times.160
.mu.m beam pattern can have an excitation laser powered at 300 mW
without increasing the total amount of power at the incident point.
Alternately, the excitation laser can be run at 150 mW to take
advantage of the lower per unit area irradiation energy, decreased
irradiation damage, longer laser life, and the like.
[0110] In comparison to conventional beam shaping optics and
conventional photomultiplier tube amplification devices, the
reduced height beam pattern optics invention and the
photomultiplier tube amplification invention can increase the
purity of X-chromosome bearing and Y-chromosome bearing populations
of spermatozoa by about 4%, or more.
[0111] The beam shaping optics invention (30) can be installed to a
flow cytometer as shown in FIGS. 15 and 16. As can be understood,
the light emitted (31) by laser excitation of fluorochrome(s) bound
to the DNA contained within spermatozoa can be detected by
photomultiplier tubes (32) situated at 0 and 90 degrees relative to
the flat surface of the sperm head (28) as it flows through the
excitation laser beam pattern.
[0112] As can be understood, stained spermatozoa must be pumped
through the excitation beam or irradiation beam in a precise manner
so that each sperm head is oriented with the flat surface of the
sperm head directed toward the photomultiplier tube that is the 0
degree detector. Accurate measurement of the DNA content of the
spermatozoa can only be measured from the flat surface of the
paddle-shaped sperm head (28). Thus, only that proportion of the
spermatozoa that enter the excitation beam in the proper
orientation can be measured accurately and sorted based upon DNA
content.
[0113] Now referring to FIGS. 17, 18, and 19, particular
embodiments of the invention can also have an particle or sperm
cell orienting nozzle (33) that hydrodynamically forces the
flattened sperm head into the proper orientation as they pass in
front of the photomultiplier(s). As shown by FIG. 17, the orienting
nozzle has interior surfaces (34) that form a cone-like shape. The
internal cone gradually changes from circular at the inlet end (35)
into a highly elliptical shape near the orifice (36) where the
stream exits the tip. The orifice (36) can be circular rather than
elliptical. Thus, the internal aspect of the orienting nozzle (34)
goes from a round entrance to a narrow ellipse to a round exit
shortly before the orifice (36). This internal shape is further
clarified by the cross sections of the orienting nozzle shown by
FIG. 18.
[0114] As shown by FIGS. 19 and 21, the injection tube (37) (which
may be about 0.061 inches in diameter) can be used with the
orientation nozzle (or with a conventional nozzle) (33) which can
be beveled near the tip to form a blade (38). The flattened blade
(38) can be oriented at an angle 90 degrees from the greatest
dimension of the ellipse in the orientation nozzle (33). The
internal diameter of the injection needle can be about 0.010 inch
in diameter forming a round orifice (39) in the center of the
flattened needle tube blade (38).
[0115] In particular embodiments of the beveled injection tube the
beveled blade can be configured in the paddle shape illustrated by
FIG. 21. The paddle shaped beveled blade can assist in maintaining
laminar flow of the sheath fluid within the nozzle (whether
conventional nozzle or orienting nozzle). As such, the laminar flow
of liquid maintained by the paddle shaped beveled blade presents
less disruption of the objects injected into it. Spermatozoa
introduced into the laminar flow of sheath fluid maintained by an
embodiment of the injector tube invention having the paddle shaped
beveled blade allows for a 20%, 30%, 40%, 50% or even greater
increase in spermatozoa sorting rates over conventional injection
tube technology. High speed sorting of spermatozoa at rates of
about 4,000 to about 10,000 sorts of each sex per second can be
accomplished. High purity (90% or greater) of the X-chromosome
bearing and Y-chromosome bearing populations can be established at
even these high sort rates. The injector tube invention with the
beveled paddle shaped tip can be used independently of or in
combination with the other inventions described herein or other
technology such as that described in U.S. patent application Ser.
No. 09/454,488 or International Patent Application No.
PCT/US00/42350, each hereby incorporated by reference.
[0116] As shown by FIG. 21, certain embodiments of the beveled
blade injector tube invention or beveled blade paddle shape
invention can further include laminar flow enhancement grooves
(40). The laminar flow enhancement grooves (40) assist in
maintaining a laminar flow to the orifice of the injector tube.
Again, the enhanced laminar flow allows for more spermatozoa to
maintain the correct orientation in the laminar sheath fluid flow
resulting in higher numbers of sortable event rates which in turn
leads to higher sort rates for each sex or spermatozoa.
[0117] In another embodiment of the invention, the orienting nozzle
orifice (39) or other conventional can be sized to form droplets
which encapsulate intact live sperm as they exit the orifice (39).
Encapsulation of the sperm cells does not occur in conventional
sperm cell entrainment technology. Rather a portion of the sperm
cell tail resides outside of the droplet. For example, bovine sperm
cells have a length of about 13.5 microseconds when the fluid
stream has a pressure of about 50 pounds per square inch (i.e. the
length of time for the entire length of the sperm cell to pass
through the irradiation beam at about 50 pounds per square inch
fluid stream pressure). Conventional droplet formation techniques
for entraining bovine sperm cells establish various conditions such
as a 14 microsecond droplet (i.e. the time it takes to form a
single droplet waveform in a fluid stream), a nozzle having an
orifice with a diameter of about 70 micrometers, and an oscillator
operated at about 35 kilohertz. Regardless of parameters selected
in conventional systems, a portion of the sperm cell tail readily
protrudes from the droplet. To prevent the sperm cell tail from
protruding from the droplet, one embodiment of the droplet
encapsulation invention provides an orifice of about 100
micrometers that can produce a droplet of about 28 microseconds at
about 50 pounds per square inch at about 30 kilohertz. By entirely
encapsulating the intact live sperm cell, including the tail
portion, the sperm cell interacts with the nozzle less upon
charging of the droplet and the deflection of the droplet can be
more accurate. This leads to less cross contamination of
X-chromosome bearing sperm with Y-chromosome bearing sperm and also
allows deflected spermatozoa to be more uniformly collected. Sperm
that are uniformly deflected can be directed to collection surfaces
that are cushioned by various liquids. Cushioning the separated
spermatozoa can be important in reducing stress as described in
U.S. patent application Ser. No. 09/001,394, hereby incorporated by
reference. With respect to spermatozoa from other species of
mammals, the invention can be varied to produce droplet sizes to
encapsulate the varying lengths of sperm cells. Depending on the
length of the spermatozoa and the pressure of the fluid stream the
droplet encapsulation invention can still achieve droplet formation
rates of at least 10,000 droplets per second, at least 20,000
droplets per second, at least 30,000 droplets per second, at least
40,000 droplets per second, at least 50,000 droplets per second, at
least 60,000 droplets per second, at least 70,000 droplets per
second, at least 80,000 droplets per second, at least 90,000
droplets per second, at least 100,000 droplets per second and so on
up to about 200,000 droplets per second in some embodiments of the
droplet encapsulation invention.
[0118] Even with the orienting nozzle invention there will be a
certain number of spermatozoa, or particles, which are not properly
oriented in the beam pattern. As described above, if the
orientation of a sperm head is not proper then the DNA content
cannot be measured accurately based upon the emitted light.
Particular embodiments of the present invention provide for the
removal of undesired unoriented spermatozoa (RUUS) or particles
within a fluid stream.
[0119] Referring now to FIGS. 16 and 20A, it can be understood that
accurate measurement of the DNA content of a spermatozoa depends
upon the flat surface of the paddle-shaped sperm head (28) being
oriented properly with the detector. Thus, only that proportion of
the spermatozoa that enter the excitation beam in the proper
orientation as shown by FIGS. 16 and 20A can be measured accurately
and sorted in to X-chromosome bearing and Y-chromosome bearing
populations based upon DNA content. As shown by FIGS. 20A and 20B,
spermatozoa which transit through the excitation beam in proper
orientation generate an oriented emission signal plot (40) that can
be shaped differently than the unoriented emission signal plot (41)
that is generated by unoriented spermatozoa shown by FIG. 20D.
Naturally, the shape of the unoriented emission signal plot (41)
generated by unoriented spermatozoa will vary depending on the
degree of improper orientation in the excitation beam. These
improper orientations can include the orientation shown in FIG. 20C
but can also include all manner of orientations that rotate the
sperm head any portion of a rotation that orients the surface of
the paddle-shaped head out of alignment with the detector (proper
alignment shown by FIG. 16), or any portion of a rotation that
orients the axis of the sperm head (42) out of alignment with the
direction of flow. Naturally, proper orientation may be defined
differently from species to species. For some species, in which the
sperm head is not paddle-shaped, the proper orientation within the
excitation beam, or relative to the detectors or otherwise, may be
defined by other anatomical characteristics or signal
characteristics. Nonetheless, an optimized signal for the properly
oriented spermatozoa of various species within the excitation
window can be generated as the standard emission signal plots for
subsequent comparison with serial emission events.
[0120] By comparing the shape (or the integrated area or both) of
each emission signal plot with the standard emission signal plot
(or standard integrated area or both) established for an oriented
spermatozoa of a species of mammal, unoriented sperm can be
identified, the signal subtracted from univariate or bivariate
histograms, and the unoriented sperm can be affirmatively removed,
if desired, so that unoriented sperm are not collected into either
the X-chromosome bearing population or the Y-chromosome bearing
population.
[0121] Importantly, as the invention(s) improve(s) resolution
between the two spermatozoa populations being separated which
increase the rate at which the populations can be separated from
one another, and improves the purity of the populations that are
separated. As such, it is now possible to sort spermatozoa at
remarkably high speeds. Sortable or separable event rates can be as
high as about 35,000 per second (not including coincident
events--multiple spermatozoa within the excitation/detection window
at the same time). Sortable or separable event rates correlate with
high separation or sort rates which can be about 5000 to about
11,000 intact live sperm of each sex per second with a purity of
90%, 92%, 93%, 95%, or greater. The above-described inventions also
allow for even higher purity X-chromosome bearing and Y-chromosome
bearing populations to be obtained of about 97% to about 98% or
even higher by reducing the sort or separation rates to around 2000
live sperm of each sex per second.
[0122] As can be understood, the above inventions described are
particularly important in achieving the highest possible sortable
or separable event rates and highest possible resulting separation
rates which can be at least 1,000 separations per second, at least
2,000 separations per second, at least 3,000 separations per
second, at least 4,000 separations per second, at least 5,000
separations per second, at least 6,000 separations per second, at
least 7,000 separations per second, at least 8,000 separations per
second, at least 9,000 separations per second, or at least 10,000
separations per second of each sex per second, or greater.
[0123] The invention allows for high speed sorting, as set forth
above, of spermatozoa even when they are difficult to stain, or
have other anatomical or chemical features, that make
differentiation between the X-bearing chromosome and Y-bearing
chromosome populations more difficult. Even in these difficult
cases, high purity X-chromosome bearing and Y-chromosome bearing
populations of bovine spermatozoa can be isolated at high purity of
92% to 93% by achieving sortable event rates of about 15,000-20,000
sortable events per second or higher as described above, and sort
or separation rates of intact live spermatozoa of each sex
(X-chromosome bearing and Y-chromosome bearing) of 2000 intact live
sperm of each sex per second.
[0124] Now referring to FIGS. 23 and 24, an embodiment of the
invention utilizes differential interference contrast technology to
measure the volume of a particle or capsule. A basic embodiment of
the invention can comprise particles that have a difference volume,
such as sperm cell heads (28) that have a difference in volume
between X-chromosome bearing and Y-chromosome bearing sperm cells.
An electromagnetic radiation source (43) generates electromagnetic
radiation or a beam of electromagnetic radiation (44) having
initial waveform characteristics differentially responsive to the
difference in volume between the particles or sperm cell heads
(28). The electromagnetic radiation which can be laser light, but
could also be numerous types of electromagnetic radiation
including, but not limited to, microwave radiation, ultraviolet
radiation, or the like. Upon traversing the particle or capsule or
sperm head volume containing phase shifting material the
electromagnetic radiation can be focused through an objective lens
(45) onto a detector (46) responsive to the waveform
characteristics of the electromagnetic radiation. The detector can
be coupled to an analyzer (47). The analyzer can differentiate
between particles based on the change in the waveform
characteristics prior to traversing the volume of the particle and
after traversing the volume of the particle and can analyze the
signal based on integrated areas or signal shape or both. In
certain embodiments the invention analyzing waveform
characteristics can comprise superimposing initial waveform
characteristics with altered waveform characteristics upon
traversing the volume of the particle, capsule, or sperm cell head.
Superimposing the initial waveform characteristics and the phase
shifted waveform characteristic can differentially modulate the
intensity of the beam of electromagnetic radiation in manner that
correlates to the amount of phase shift media the electromagnetic
radiation traverses. The invention may also include additional
filters (48), such as color filters.
[0125] Now referring to FIG. 24, an embodiment of the optics
invention involves using differential interference contrast optics
that increase the actual distance over which the light is split up
compared to conventional DIC microscopy which corresponds to the
resolution limit of the microscope. In this embodiment of the
invention, the induced split is larger than the size of the
objects, thus giving rise to two individual images, separated
laterally, originating from one object. The second modification
involves using plates of birefringent material, such as Savart
plates, at a location away from the objective lens. This embodiment
of the invention is easier to construct since the birefringent
materials do not have to be located inside the objective housing.
In conventional DIC microscopes the birefringent material is used
in the form of so-called Wollaston prisms, that have to be located
inside the objective housing, making it necessary to use expensive
objective lenses that have been manufactured specifically for this
purpose.
[0126] Components of an embodiment of the invention may be arranged
in line with each other and consist of: a source of electromagnetic
radiation (43), for example, a Mercury arc lamp; a spectral
adjustment element, for example, a bandpass filter; a polarization
adjustment element (49), for example, a sheet polarizer (53) and a
waveplate (54) responsive to a rotatable mount; a light condenser
(51) allowing the light to be condensed onto the particle or sperm
cell, for example, a condenser lens, or set of lenses, or
microscope objective; a fluid stream (8) that may contain particles
or sperm cells (28), for example a fluid jet ejected under
pressure; a light collector (45) to collect the light from the
particle or cell, for example a 50.times. high working distance
microscope objective and a tube lens; a beam splitter (50) to split
up the beam into two, or more, components, for example, a piece of
birefringent material in the form of a Savart plate, mounted in
such a way that its orientation and location can be controlled
accurately; image light selector (55) to select only the light
corresponding to the particle or sperm cell, for instance a set of
pinholes, one pinhole (53) for each of the images formed.
[0127] In one embodiment of the invention, the components may be
arranged in such a way that the light source (43) or its image are
located at the back focal plane of the light condenser (45) often
referred to as Kohler type illumination. The image of the object
plane may best coincide with the object light selector (55) or
pinhole(s) (53), in order to capture the light from individual
particles or sperm cells. As shown in FIGS. 27 and 28, components
can be mounted on a sturdy optical table, or bench, using
mountings, posts, and holders. Components can be mounted in such a
way that focusing of the object plane can be done accurately. This
can be done by equipping the fluid stream with a stream position
controller, such as micrometers, in order to turn the stream in and
out of focus. In addition it may be necessary to equip the light
condenser (51) with a light condenser position controller (61)
allowing it to be focused onto the object plane. It may be
necessary to take special care about the mounting of the
birefringent elements or beam splitter (50), a three axis rotation
element may be preferable.
[0128] Now referring to FIG. 25, embodiments of the present
invention may also include the use of both generated images, in
order to determine the orientation of an asymmetrical particles the
fluid stream, including, but not limited to, spermatozoa such as
bull sperm cells. An orientation assessment embodiments of the
invention can include an optical system that allows for control of
the polarization state of the light entering the system for both
generated images independently. The interference optics invention
may further provide polarization adjustment element (56) that
controls the polarization state of light entering the system. For
the orientation detection invention the polarization adjustment
element (56) may be selected in such a way that it consists of two
parts, that are imaged onto image light selector (55) that in one
embodiment of the invention contains the pinholes (53). This can be
accomplished by locating the polarization adjustment element (56)
in the conjugate plane of the image plane (55), or by using other
optics, to accomplish the same thing. A simple example of this
component may be a `half-shade` piece, for instance consisting of
two hemi-circular parts of polarizing material, such as
sheet-polarizer, the orientation angle of which may be chosen
independently. Each pinhole in the image plane can fall in one of
the halves of said hemisphere. The polarization angles can be
chosen in such a way that the signal of one pinhole (53)
corresponds to the volume, and is relatively independent from the
orientation angle of the passing object, and the other pinhole (53)
has a signal that depends, to a great degree, upon this orientation
angle. The two signals may be processed by analyzer (47) in a
manner similar to a conventional multi-channel flow cytometry, as
but one example. With respect to this example, bivariate dotplots
can be made, and also allow the user to select windows on this
plot.
[0129] An improvement of the `half-shade` piece described above may
be the construction shown by FIG. 25D. The same said two
hemispherical parts are projected onto the image plane but the way
they are generated is different. A mirror (57) breaks up the light
(44) into the hemi-circular parts, and recombines them back to
back. Each of the halves traverses a separate means to control its
polarization state. An advantage of this embodiment is that the
polarization angles can be controlled continuously and
independently, thus facilitating the adjustment of the set-up.
Materials used in this embodiment can be supplied by standard
optical supply firms, and can be mounted in the set-up using
similar mounting materials as used for the interferometric
optics.
[0130] Now referring to FIG. 26, In order to correct for artifacts
introduced by having light pass through a non-flat region of
transparent material, such as a substantially cylindrical fluid
stream but including other geometries as well, embodiments of the
present invention disclose the incorporation of a component similar
in shape to the non-flat region, but opposite in terms of relative
refractive indices. In the specific case of a flow cytometer this
shape approximates a cylinder. To correct for artifacts introduced
by the fact that the objects to be assessed are located within a
cylindrical stream of water, is the incorporation of an optical
component (58) which can be in the shape of a transparent cylinder,
located inside transparent material (59) of a higher refractive
index. It may be preferred that the image of the stream and of the
compensation element fall on top of each other in the image plane.
This can be done by locating the compensation element between the
objective lens and the image plane, and by incorporating auxiliary
lenses.
[0131] An embodiment of the optical component (58) can be located
within a thin slice of transparent material of higher refractive
index (59), for instance glass, or Perspex.TM., with a cylindrical
hole drilled across it. Perspex' has the advantage that it can be
easier to drill a round channel into it. The cylindrical hole may
be filled by a transparent material, the refractive index of which
is lower than that of the surrounding material. The difference in
refractive index between the substance and the surrounding material
can be the same as but opposite to the difference in refractive
index between the water in the stream and the surrounding air for
certain applications. It may not be necessary to have the cylinder
the same size as the stream of water, as long as magnification by
the lenses used, makes the resulting images in the image plane the
same size. In some applications, it may be desired or necessary to
adjust the refractive index difference to compensate for this
magnification. Manufacturing of such element out of Perspex.TM. can
be quite simple, and can be done by most mechanical workshops that
have experience with machining Perspex.TM. or the selected
material. It may be made in such dimensions that it fits in a
standard optical mounting hardware, to facilitate incorporation
into the optics.
[0132] Exactly matching the refractive indices may be difficult. An
embodiment of the invention that facilitates adjustment can be to
make the substance inside the Perspex.TM., or other selected
material, a transparent refractive index fluid (58), as but one
example, an organic oil, or mixture of oils that have a refractive
index close to the desired one. Due to the fact that the refractive
index of most fluids changes with temperature, much more so than
solids, or glasses, it may be possible to fine-tune the difference
in refractive index by temperature. This may be done by
incorporating a temperature controller (60).
[0133] Optical component (58) of transparent fluids or refractive
index fluids can be supplied by chemical supply firms. These firms
often have data regarding the refractive index of their fluids
readily available. Some firms even offer fluids that are specially
made to serve as refractive index fluids, and have a guaranteed and
stable refractive index. Temperature controllers and thermostats
are supplied by many firms. A practical way to apply heat to the
refractive index fluid can be to use a hollow mounting made of heat
conducting material, a metal as but one example, containing the
refractive index fluid. Using a conventional immersion thermostat
cycler, found in many laboratories, water can be pumped through the
mounting, thus keeping the element at a fixed and controllable
temperature.
[0134] 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
functionally-oriented terminology, each aspect of the function is
accomplished by a device, subroutine, or program. Apparatus claims
may not only be included for the devices 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 which
now be included.
[0135] 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 "droplet separator"
should be understood to encompass disclosure of the act of
"separating droplets"--whether explicitly discussed or not--and,
conversely, were there only disclosure of the act of "converting
liquid-gas", such a disclosure should be understood to encompass
disclosure of a "droplet separator" and even a means for
"separating droplets". Such changes and alternative terms are to be
understood to be explicitly included in the description.
[0136] Additionally, the various combinations and permutations of
all elements or applications can be created and presented. All can
be done to optimize the design or performance in a specific
application.
[0137] 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. Specifically, U.S. Patent Application
Nos. 60/267,571, 60/239,752, and 60/203,089 are each hereby
incorporated by reference herein including any figures or
attachments, and each of references in the following table of
references are hereby incorporated by reference.
[0138] 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. 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).
[0139] In addition, 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 in countries such as Australia and the like.
[0140] Thus, the applicant(s) should be understood to have support
to claim at least: i) each of the devices described herein, ii) the
related methods disclosed and described, iii) similar, equivalent,
and even implicit variations of each of these devices and methods,
iv) those alternative designs which accomplish each of the
functions shown as are disclosed and described, v) those
alternative designs and methods which accomplish each of the
functions shown as are implicit to accomplish that which is
disclosed and described, vi) each feature, component, and step
shown as separate and independent inventions, vii) the applications
enhanced by the various systems or components disclosed, viii) the
resulting products produced by such systems or components, ix)
methods and apparatuses substantially as described hereinbefore and
with reference to any of the accompanying examples, and the x) the
various combinations and permutations of each of the elements
disclosed.
[0141] In addition, 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 in countries such as Australia and the like.
[0142] The claims set forth in this specification by are hereby
incorporated by reference as part of this description of the
invention, and the applicant expressly reserves the right to use
all of or a portion of such incorporated content of such claims as
additional description to support any of or all of the claims or
any element or component thereof, and the applicant further
expressly reserves the right to move any portion of or all of the
incorporated content of such claims or any element or component
thereof from the description into the claims or vice-versa as
necessary to define the matter for which protection is sought by
this application or by any subsequent continuation, division, or
continuation-in-part application thereof, or to obtain any benefit
of, reduction in fees pursuant to, or to comply with the patent
laws, rules, or regulations of any country or treaty, and such
content incorporated by reference shall survive during the entire
pendency of this application including any subsequent continuation,
division, or continuation-in-part application thereof or any
reissue or extension thereon.
* * * * *