Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02858892 2014-08-11
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A PARTICLE DIFFERENTIATION APPARATUS FOR SPERMATOZOA
TECHNICAL FIELD
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.
BACKGROUND
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, United States Patent 5,135,759.
However, conventional technologies for separating spermatozoa into X-
chromosome bearing and Y-chromosome bearing populations can result in
spermatozoa
populations haying low purity. Regardless of the separation method spermatozoa
have
not been routinely separated into X-chromosome bearing and to Y-chromosome
bearing
sperri samples having high purity, such as 90%, 95%, or greater than 95%.
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 United States Patent No.
4,474,875, a
buoyant force is applied to all sperm cells simultaneously and X-chroniosome
bearing and
Y-chromosome bearing spermatozoa may then be isolated at different locations
in the
separation medium. United States Patent No. 5,514,537 discloses a technique
whereby
spermatozoa traverse a colon's' 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. United States Patent NO. 4,605,558 discloses that spermatozoa
may be
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made differentially responsive to a density gradient and United States Patent
No.4,009,260 exploits the differences in migration-rate, or swimming-speed,
between the
Y-bearing spermatozoa, and the X-chromosome hearing spermatozoa, through a
column
of retarding mcdnun.
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.
Flow cytometer techniques for the separation of spermatozoa have also been
disclosed. Using these techniques spermatozoa may be stained with a
fluorochrorne 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 parametel
chromatograms or histograms. The number of cells and fluorescence per cell may
be used
as coordinates. See United States Patent No. 5,135,759,
However, with respect to this type of technology a variety of problems remain
unresolved and isolating highly purified populations of X-chromosome bearing
orY-
chromosome bearing sperm cells be difficult.
A significant problem with conventional flow cytometer technologies can be the
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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.
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.
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
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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 30pm while the width can be approximately 80 pm. The nucleus of a
bovine
spermatozoa which contains fluorochrome bound DNA can be about 9 pm 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.
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.
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.
Another significant problem with conventional flow cytometer technologies can
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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.
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.
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, stoicbiometric 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.
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
A broad object of the invention can be to provide isolated high purity X-
chromosome
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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.
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.
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 optioal 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.
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 speini nuclei) having a graded level of high purity in the range of 80%,
85%, 90%, 95%,
or even greater than 95%.
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.
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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 eytorneter.
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.
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.
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%.
Another significant object of particular embodiments of the invention can be
to
differentiate between photoemissive events having small differences in total
emitted light
flux.
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.
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.
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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.
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.
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.
Another significant object of particular embodiments of the invention can be
to
increase the signal to background signal ratio from measured photoemissive
events.
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.
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).
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.
Another significant object of particular embodiments of the invention can be
to
increase the sperm sorting rate of flow cytometer systems.
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Another significant object of particular embodiments of the invention can be
to
increase the purity of the sperm samples sorted by flow cytometry.
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 spenn 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.
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.
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.
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.
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_
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.
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Another significant object of particular embodiments of the invention can be
to fully
encapsulate irregularly shaped objects within a drop of fluid.
Another significant object of particular embodiments of the invention can be
to
differentiate undesirably oriented objects from desirably oriented objects in
a fluid stream.
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.
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 oriented properly to allow a 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.
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
behaviour of the compensation element.
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In accordance with an aspect of the present invention, there is provided a
method
differentiating particles, comprising the steps of:
a. collecting asymmetric particles;
b. establishing said fluid stream with a flow cytometer;
c. introducing said asymmetric particles into said fluid stream;
d. orienting said asymmetric particles with respect to a detector;
e. detecting light having characteristics differentially responsive to
asymmetric
particle orientation to said detector;
f converting said light having characteristics differentially responsive to
asymmetric particle orientation into at least one signal containing asymmetric
particle
orientation information;
g. analyzing asymmetric particle orientation information; and
h. determining orientation of said asymmetric particles with respect to said
detector.
In accordance with another aspect of the present invention, there is provided
a method
differentiating sperm cells, comprising the steps of:
a. establishing a fluid stream with a flow cytometer;
b. introducing said sperm cells into said fluid stream,
c. orienting said sperm cells relative to a detector;
d. detecting light having characteristics differentially responsive to
sperm cell
orientation to said detector;
e. converting said light having characteristics differentially responsive
to sperm cell
orientation into at least one signal containing sperm cell orientation
information;
analyzing spenn cell orientation information;
comparing integrated signals generated in response to said sperm cells; and
g. determining orientation of said sperm cells relative to said
detector.
1 Oa
In accordance with another aspect of the present invention, there is provided
a method of
differentiating sperm cells, the method comprising the steps of:
a. establishing a fluid stream with a flow cytometer;
b. introducing said sperm cells into said fluid stream;
c. orienting said sperm cells relative to a detector;
d. detecting fluorescent light having characteristics differentially
responsive to sperm cell
orientation to said detector;
e. converting said fluorescent light having characteristics differentially
responsive to
sperm cell orientation into at least one signal containing sperm cell
orientation information;
f. analyzing the sperm cell orientation information;
g. comparing integrated signals generated in response to said sperm cells to
integrated
signals generated in response to oriented sperm cells; and
h. determining orientation of said sperm cells relative to said detector.
In accordance with another aspect of the present invention, there is provided
a method of
differentiating sperm cells, the method comprising the steps of:
a. establishing a fluid stream with a flow cytometer;
b. introducing said sperm cells into said fluid stream;
c. orienting said sperm cells relative to a detector;
d. detecting fluorescent light having characteristics differentially
responsive to sperm cell
orientation to said detector;
e. converting said fluorescent light having characteristics differentially
responsive to
sperm cell orientation into at least one signal containing sperm cell
orientation information;
f. analyzing the at least one signal containing the sperm cell orientation
information;
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g. comparing integrated signals generated in response to the at least one
signal containing
sperm cell orientation information for non-orientated sperm cells to
integrated signals generated
in response to the at least one signal containing sperm cell orientation
information for oriented
sperm cells; and
h. determining orientation of said sperm cells relative to said detector based
on the
comparison of the integrated signals of the step (g).
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Naturally further objects of the invention are disclosed throughout other
areas of the
specification and claims.
TV. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a generalized flow cytometer.
Figure 2 shows a second view of a generalized flow cytometer.
Figure 3 shows a comparison of univariate histograms from flow cytometers (#1,
#2,
and #3) without the amplifier invention (Figure 3A) with univariate histograms
for the same
flow cytometers using a particular embodiment of the amplification invention
(Figure 3B)
illustrating the improved resolution between X-chromosome bearing and Y-
chromosome
bearing populations of bovine spermatozoa.
Figure 4 shows univariate and bivariate histograms illustrating the
conventional
resolution between X-chromosome bearing and Y-chromosome bearing populations
of
bovine spentiatozo a.
Figure 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.
Figure 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.
Figure 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.
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Figure 8 shows univariate and bivariate histograms illustrating the
conventional
resolution between X-chromosome bearing and Y-chromosome bearing populations
of
equine spermatozoa.
Figure 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.
Figure 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.
Figure 11 shows a particular embodiment of the circuit board modification to
make
the amplification invention with respect to a MoFle flow cytometer.
Figure 12 shows an electrical schematic diagram of a particular embodiment of
the
amplification invention with respect to a MoFlo flow cytometer.
Figure 13 shows the laser beam pattern using conventional beam shape optics
(Figure
13A) and the laser beam pattern using the reduced height beam shape optics
(Figure 13B).
Figure 14 shows a bar graph that compares the purity of separated X-chromosome
bearing spermatozoa (Figure 14A) and Y-chromosome bearing spermatozoa (Figure
14B)
using conventional technology or using the amplification invention
independently or in
conjunction with reduced height beam shaping optics.
Figure 15 shows a front view of the reduced height beam shaping optics.
Figure 16 shows a top view of the reduced height beam shaping optics.
Figure 17 shows a perspective and two cross sections of the object orienting
nozzle
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invention.
Figure 18 shows a graded series of cross sections of the object orienting
nozzle
invention.
Figure 19 shows a front view and an end view of an embodiment of the beveled
injection tube invention.
Figure 20 illustrates the removal of undesired unoriented spermatozoa (RUUS)
invention by comparison of signal(s) from the oriented spermatozoa (Figures
20A and 20B)
and the signal(s) from the unoriented spermatozoa (Figures 20C and 20D).
Figure 21 shows a perspective of another embodiment of the beveled injection
tube
invention having a paddle shaped beveled blade.
Figure 22 shows a conventional optics technology coupled to a flow cytometer.
Figure 23A shows the shape and size of a typical spermatozoon and Figure 23B
shows the difference between correctly and non-correctly orientated
spermatozoa.
Figure 24 shows an embodiment of the invention having construction allowing
the
measurement of two signals, for example volume and orientation.
Figure 25A and B shows an embodiment of the invention having two halves with a
pinhole corresponding to each half, Figure 25C shows an image plane of an
embodiment of
the invention, Figure 25D shows an embodiment of the invention having two
independently
rotatable polarizers.
Figures 26A and 26B illustrates the compensation method for the fluid stream
for an
embodiment of the invention, Figure 26C shows an embodiment of a compensation
element,
26 D shows another embodiment of a compensation element where images of a
fluid stream
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and from the compensation element fall on top of each other in the image
plane.
Figure 27 shows an embodiment of the interference optics invention.
Figure 28 shows an a second view of the interference optics invention.
V. MODE(S) FOR CARRYING OUT THE INVENTION
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- iable 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.
High purity separated spermatozoa from the various species of mammals can be
incorporated into products that can be used with artificial insemmation
protocols or as part of
commercial business methods such as those as described in United States Patent
Application
Nos. 60/211,093, 60/224,050, or WO 2000/06193; or be used with low dose
insemination
protocols as described in WO 1999/33956, or used in vitro fertilization of
oocytes
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from animals, including humans, as described in United States Patent
Application No.
60/253,785,
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,
a 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 1.00%.
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_
Moreover, while this disclosure provides descriptions of embodiments of
apparatus and methods for flow separation of X-chromosome bearing spermatozoa
from
CA 02858892 2014-08-11
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 mariner so that they may be applied to the wide variety of
applications.
Now referring to Figures 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).
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 enviromn.ent, 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.
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
16
CA 02858892 2014-08-11
(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
dependant upon the amount of DNA contained within each spermatozoa. Because X-
chromosome bearing spermatozoa contain more DNA than Y-chromosome bearing
spermatozoa, the X-chromosome bearing spermatozoa can bind a greater amount of
fluorochrome than Y-chromosome bearing spermatozoa. Thus, by measuring the
fluorescence emitted by the bound fluorochrome upon excitation, it is possible
to
differentiate between X-bearing spermatozoa and Y-bearing spermatozoa.
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.
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
17
CA 02858892 2014-08-11
discussed in United States Patent Application 09/001,394. Naturally, numerous
deflection
trajectories can be established and collected simultaneously.
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.
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.
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.
18
CA 02858892 2014-08-11
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.
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 United States Patent 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.
Unexpectedly, reducing the amount of voltage to the photomultiplier tube below
400 volts to about 280 volts, or about 250 volts, or even to 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
19
CA 02858892 2014-08-11
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.
As a specific example, existing Cytomation SX MoFlo sorting flow eytometers
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 flow cytometers can be converted by reconfiguring the photomultiplier
controllers.
The R16C resistor (2.49 kilohms) on channel three can be replaced by a 2.0K
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
flow cytometers with two 3.75 kilohm resistors in parallel, or a 1.3 kilohm
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.
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.
Now referring to Figure 3, a comparison of univariate histograms generated on
three
different SX MoFlo flow cytometers (#1, #2, #3) prior to the use of the
invention (Figure
3A), and using the invention (Figure 3B) 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
CA 02858892 2014-08-11
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.
The mean separation rate or sort rates of intact live spermatozoa prior to use
of
this embodiment of the invention with the SX Mono flow cytometers was about
17.9 x
106/4.5 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.
The mean sort rates of live spermatozoa after the above-mentioned conversion
was
about 40_3 x 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.
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.
Similarly, referring now to Figures 4 and 5, which show bivariate histograms
from sorting of intact live bull spermatozoa with the SX MoFle flow cytometer
i11 prior
to using the invention (Figure 4) and after the above-mentioned conversion
(Figure 5).
Prior to using the invention, the SX MoFlo flow cytometer was initially
operated at 440
volts at the photocathode with the laser adjusted to 135 MW, a gain of 1X 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 flow cytometer was operated at about 262
volts at the
photocathode, with the laser adjusted to about 100mW, a gain of 4X, 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
21
CA 02858892 2014-08-11
valley between the X-chromosome bearing population (19) and the Y-chromosome
bearing population (20).
Similarly, referring now to Figures 6 and 7, which show bivariate histograms
from
sorting of intact live bull spennatozoa with the SX MoFlo flow eytometer #2
before
using this embodiment of the invention (Figure 6) and upon using this
embodiment of the
invention (Figure 7) operated at the same parameters as shown in Figures 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).
Now referring to Figures 8 and 9, which show bivariate histograms from
separation or sorting of intact live equine spermatozoa with the SX MoFlo
flow
cytometer before using this embodiment of the invention (Figure 8) and upon
using this
embodiment of the invention (Figure 9). When using this embodiment of the
invention,
live equine spermatozoa were separated or sorted with the laser power at 100mW
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%.
Now referring to Figure 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
22
CA 02858892 2014-08-11
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 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 speini nuclei are nearly fully resolved to
baseline as shown
by the univariate plot.
Now referring to Figure 11, a modification specifically for SX MoFle flow
cytometer includes the use of two resistors in parallel to provide the correct
value of 1.8K.
Two 3.57K resistors (25) and (26) are equal to about 1.785K 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.
Figure 12, provides a electrical schematic diagram for this particular
embodiment of the
invention.
Another important embodiment of the invention can be a reduced height
irradiation beam pattern optics. As shown by Figure 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 speini head(s) in the
beam pattern
(27). This results in a reduced difference in mean light flw: 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
23
CA 02858892 2014-08-11
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.
By reducing the height of the beam shape as shown by Figure 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 m, it has been
found that
the height of the beam can be about 20 p.m. In this application, it has been
found that
vertical beam heights of less than 2011m did not provide an additional gain in
resolution.
Referring to Figure 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 (Figure 14A) and sorted populations of Y-
chromosome bearing bovine sperm (Figure 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 Figure 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 photomultiplicr 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.
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
24
CA 02858892 2014-08-11
result in less stress or damage to the spermatozoa.
Again referring to Figure 14B, it can be understood that the reduced height
beam
pattern can be used in conjunction with a irradiation beam pattern having
greater area than
conventionally used. For example, conventional beam patterns (27), such as
that shown
in Figure 14A, have an elliptical pattern of about 30 um X 80 urn 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 um X 160
urn
beam pattern (29). The 20 um X 160 urn beam pattern has approximately 1.3
times the
area of the 30 urn X 80 urn 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 urn X
80um irradiation beam pattern and the excitation laser is conventionally
powered at
150mW, then particular embodiments of the invention with a 20 urn X 160 urn
beam
pattern can have an excitation laser powered at 300mW without increasing the
total
amount of power at the incident point. Alternately, the excitation laser can
be run at
150mW to take advantage of the lower per unit area irradiation energy,
decreased
irradiation damage, longer laser life, and the like.
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.
The beam shaping optics invention (30) can be installed to a flow cytometer as
shown in Figures 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.
CA 02858892 2014-08-11
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.
Now referring to Figures 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 Figure 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 Figure 18.
As shown by Figure 19 and 21, the injection tubc (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 rounded orifice (39) in the center of the
flattened needle
tube blade (38).
In particular embodiments of the beveled injection tube the beveled blade can
be
configured in the paddle shape illustrated by Figure 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
26
CA 02858892 2014-08-11
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%o, 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. 6,263,745
or WO
2001/40765.
As shown by Figure 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 cozect 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.
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 spezn 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 imadiation beam at about 50 pounds per square
inch
fluid stream pressure). Conventional droplet formation techniques for
entraining
bovine sperm cells establish a 14 microsecond droplet (i.e. the time it takes
to
form a single droplet waveform in a fluid stream) from a nozzle having an
27
CA 02858892 2014-08-11
orifice with a diameter of about 70 micrometers which can be made responsive
to an
oscillator operated at about 35 kilohertz. As such, a portion of the sperm
cell tail readily
protrudes from the droplet. To prevent the spenn 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. Spean 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. 6,149,867. 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.
Even with the orienting nozzle invention theie 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 unonented spermatozoa (RUUS) or
particles
within a fluid stream.
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CA 02858892 2014-08-11
Referring now to Figures 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 Figures 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 Figures 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 Figure 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 Figure 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
Figure 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.
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.
29
CA 02858892 2014-08-11
Importantly, as the invention(s) improve(s) resolution between the two
spermatozoa
populations being separated which increases 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 separate
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
separate 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.
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.
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-bcaring 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.
CA 02858892 2014-08-11
Now referring to Figures 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.
Now referring to Figure 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
31
CA 02858892 2014-08-11
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.
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 50x 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 fauii 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.
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
Figures 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
32
CA 02858892 2014-08-11
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.
Now referring to Figure 25, embodiments of the present invention may also
include the use of both generated images, in order to determine the
orientation of a
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 clement (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.
An improvement of the 'half-shade' piece described above may be the
33
CA 02858892 2014-08-11
construction shown by Figure 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.
Now referring to Figure 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.
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, 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
34
CA 02858892 2014-08-11
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 can be quite simple, and can be
done by
most mechanical workshops that have experience with machining perspex 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.
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, 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).
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.
The discussion included in this PCT 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
CA 02858892 2014-08-11
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.
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.
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
36
CA 02858892 2014-08-11
,
,
performance in a specific application.
..
= ______________________________________________________ -i- .
DOCUMENT NO. DATE NA/Y1E CLASS SUBCLASS F1LING DATE
32,350 02/10/87 Bhattacharya 204 180.1
11/22/74
__________________________________________________________________ _
3,687,806 08/29/72 Van den BoYenkamp 195 1.3 I 1/04/69
3,829,216 08/13/74 Persidsky 356 36 10/02/72
_ _________________________________________________________________
3,894,529 07/15/75 Shnmpton 128 1 R 04/10/69
4,009,260 02/22/77 Ericcson 424 561 10/11/74
4,067,965 01/10/78 Bhattacharya 424 105 12/17/75
4,083,957 04/11/78 Lang 424 78 02/04/76
4,085,205 04/18/78 Hancock 424 105 01/24/77
________________________________________________________ ._ _____
________________ , ____
4,092,229 05/30/78 Bhattachaiya 204 180 R 10/20/76
4,155,831 05/22/79 Bhattacharya 207 299 R 02/23/73
__________________________________________________________________ 1
4,191,749 03/04/80 Bryant 424 105 10/11/77
4,225,405 09/30/80 Lawson 204 180 R 08/16/78
4,276,139 06/30/81 Lawson 204 180 R 10/09/39
1 4,339,434 07/13/82 Enccson 424 561 08/17/81
4,362,246 12/07/82 Adan 209 3.3 07/14/80
______________________________________ _ __
4,448,367 05/15/84 Bryant 424 .85 02/15/80
4,474,875 10/02/84 Ski !raptor! 435 002 08/18/80
______________________________________ _
4,501,366 02/26/85 Thompson 209 556 12/14/82
4,511,661 04/16/85 Goldberg 436 503 12/30/83
4,605,558 08/12/86 Shrimpton 424 561 04/20/84
4,660,911 04/28/87 Sage at al 356 39 05/03/84
4,680,258 07/14/87 Hammerhng et al 435 7 08/09/83
4,698,142 10/06/87 Moroi et al 204 ' 182.3
07/31/85
4,749,458 06/07/88 Moroi eta! 204 182.3 03/02/87
4,988,619 01/29/91 Pinkel 435 30 1130/87
37
CA 02858892 2014-08-11
4,999,283 03/12/91 Zavos eta! 435 2 08/18/89
,
5,021,244 06/04/91 Spaulding 424 561 05/12/89
5,135,759 08/04/92 Johnson 424 561 04/26/91
_
5,346,990 09/13/94 Spaulding 530 350 03/12/91
. .
5,371,585 12/06/94 Morgan et al. 356 246 11/10/92
5,439,362 08/08/95 ' Spaulding 424 185.1 07/25/94
5,466,572 11/14/95 Sasaki etal. 435 2 04/25/94
5,483,469 01109/96 Van den Engh et al. 364 555 08/02/93
5,503,994 04/02/96 Shear et al. 436 90 10/08/93
5,514,537 05/07/96 Chandler 435 002 11/28/94
5,589,457 12/31/96 Wiltbank 514 12 07-03-95
5,602,039 02/11/97 Van den Engh 436 164 10/14/94
5,602,349 02/11/97 Van den Engh 73 864.85 10/14/94
5,660,997 08/26/97 Spaulding 435 7.21 06/07/95
5,690,895 11/25/97 Matsumoto etal. 422 73 12/06/96
_
5,700,692 12/23/97 Sweet 436 50 09/27/94
5,726,364 03/10/98 Van den Engh 73 864.85 02/10/97
5,819,948 10/13/98 Van den Engh 209 158 08/21/97
5,880,457 03/09/99 Tomiyama et al. 250 207 06/16/97
5,985,216 11/16/99 Rena, etal. 422 073 07/24/97
6,071,689 06/06/00 Seidel etal. 435 2 01/29/98
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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
49
CA 02858892 2014-08-11
should be understood for each term and all definitions, alternative terms, and
synonyms such
as contained in the Random House Webster's Unabridged Dictionary, second
edition.
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.
Thus, the applicant(s) should be understood to have support to claim at least:
I) each
of the liquid to gas conversion 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.
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 hut not
the exclusion of
any other element or step or group of elements or steps.
