HK1163810B - A flow cytometer - Google Patents
A flow cytometer Download PDFInfo
- Publication number
- HK1163810B HK1163810B HK12104128.3A HK12104128A HK1163810B HK 1163810 B HK1163810 B HK 1163810B HK 12104128 A HK12104128 A HK 12104128A HK 1163810 B HK1163810 B HK 1163810B
- Authority
- HK
- Hong Kong
- Prior art keywords
- sperm
- flow cytometer
- chromosome bearing
- sperm cell
- cells
- Prior art date
Links
Description
The application is a case-splitting application again, the Chinese national application number of the original case-splitting application is 200810166001.8, the application date is 5, 9 and 2001, and the invention name is 'sperm group with high-purity X-chromosome and Y-chromosome'; the original application has the international application number of PCT/US01/15150, the Chinese national application number of 01810748.6, the application date of 5-9.2001, and the invention name of the invention is 'sperm population with high-purity X-chromosome and Y-chromosome'.
Technical Field
Isolated populations of sperm having high purity X-chromosomes or Y-chromosomes and methods of separating sperm, particles, or events according to distinguishing characteristics such as mass, volume, DNA content, and the like.
Background
Isolated populations of spermatozoa having high purity X-chromosomes or Y-chromosomes can be used to perform in vivo or in vitro fertilization or artificial insemination of oocytes or oocytes of many mammals, such as bovines, equines, ovines, caprines, porcines, dogs, cats, camels, elephants, bulls, buffalo, and the like. See also, U.S. Pat. No.5,135,759, incorporated herein by reference.
However, conventional methods of separating populations of X-chromosome bearing and Y-chromosome bearing sperm will produce populations of sperm of lower purity. Regardless of the method, sperm has not been separated into sperm samples having, for example, 90%, 95%, or greater than 95% purity of either X-chromosome or Y-chromosome.
Several methods have been disclosed for separating Y-chromosome bearing sperm from X-chromosome bearing sperm, either directly or indirectly based on size, mass, or intensity differences. As disclosed in U.S. Pat. No.4,474,875, a buoyancy force is applied simultaneously to all sperm cells to separate X-chromosome bearing or Y-chromosome bearing sperm in the separation medium into distinct locations. U.S. Pat. No.5,514,537 discloses a technique in which sperm is passed through a chromatography column containing two sizes of beads. Sperm with a large X-chromosome are separated into layers containing large beads, while sperm with a smaller Y-chromosome are separated into layers containing smaller beads. U.S. patent No.4,605,558 discloses a method by which sperm cells can be made differentially sensitive to density gradients. While U.S. patent No.4,009,260 utilizes the difference in migration or motility rates of X-chromosome bearing sperm and Y-chromosome bearing sperm in a barrier medium passing through a column.
A problem common to each of the above-mentioned techniques is that they work in "bulk" on sperm, meaning that all sperm cells are subjected to the same treatment at the same time, and sperm cells bearing the Y-chromosome emerge faster, earlier, or at a different location than sperm cells bearing the X-chromosome. Thus, individual sperm cells cannot be identified and there is no actual "measure" of volume, mass, density or other sperm cell characteristic. The benefits of individually identifying sperm cells in an actual separation process can be detected and, even during the separation process, physical quantitative data can be generated and separation parameters varied as desired. These techniques cannot be used with flow cell screening devices.
Flow cytometry techniques for separating sperm have also been disclosed. Using these techniques, sperm cells should be stained with a fluorescent stain and flowed through a narrow stream or band of excitation or illumination, such as a laser beam. The fluorescent stain fluoresces when the stained particles or cells pass through an excitation or illumination source. The fluorescence can be collected by optical means, focused on a detector like a photomultiplier tube capable of generating and multiplying an electrical signal, and then analyzed by an analyzer. These data are then displayed using a multi-parameter or single parameter chromatogram or histogram. The number of cells and the fluorescence of each cell can be used as coordinates. See U.S. Pat. No.5,135,759, incorporated herein by reference. However, there are still a number of unsolved problems with this type of technique and it is difficult to isolate X-chromosome bearing or Y-chromosome bearing sperm cells in high purity.
One significant problem with conventional flow cytometry techniques is the orientation of the substance, particle, or cell in the sheath fluid. This is particularly problematic when the substance or cell is irregular in shape with respect to more than one axis, such as a sperm. One aspect of this problem should be to establish an initial orientation of the species in the sheath fluid. A second aspect of this problem is to maintain the orientation of the object relative to the detector (photomultiplier tube or other similar instrument) during the time that the substance being measured is emitting light.
Another significant problem with conventional flow cytometer techniques is the inability to encapsulate substances or cells in droplets. In particular, when droplets are formed around irregularly shaped objects, the size of the droplets is not sufficient to completely cover all parts of the substance or cell. For example, during flow cytometry operations, droplets as described above can be formed at very fast rates, even as much as 10,000 to 90,000 per second, and in some cases as much as 80,000 per second. When the sperm are encapsulated in the droplet, particularly at such a fast rate, a portion of the tail or neck of the sperm is not encapsulated in the droplet. The portion of the tail and neck that is not encapsulated in the droplet may react with the nozzle or the surroundings of the droplet in some way that interferes with the subsequent droplet formation or proper deflection of the droplet. Some sperm are therefore not analyzed at all, thereby reducing the efficiency of the process, or are not eliminated sufficiently to assign a value to a population of cells, or deviate from normal trajectory, or a combination of all.
Another significant problem with conventional flow cytometer techniques, as well as others, is coincidence of measurement events. One aspect of this problem is that incident flux from a first event continues to generate a signal after incident flux from a second event begins to generate a signal. Thus, at least some of the two events remain indistinguishable from each other. Another aspect of this problem is that two or more events occur simultaneously and the incident light flux includes contributions from all events. In this way, the diversity of events is not resolved at all and objects for event diversity are erroneously assigned to a sperm group or not assigned to a sperm group at all, or both. In particular, with respect to flow cytometry, suspended individual particles, objects, cells, or sperm undergo a measurable reaction, such as fluorescence, when passed through a beam of light that causes them to react with each other. In conventional flow cytometry, Hoechst stained sperm cause fluorescence emission when passed through a laser. After the actual emission event is over, the fluorescence emitted from the excited DNA-bound fluorescent agent is bright enough to produce a flow of electrons in a conventional photomultiplier tube for a period of time. Also, in conventional flow cytometers, the laser beam is capable of producing a pattern having a height of 30 μm and a width of about 80 μm. The nucleus of bovine sperm containing a fluorescent stain that binds to DNA may be about 9 μm long, making the laser beam three times greater in height than the nucleus. This difference allows the laser to simultaneously stimulate the bound fluorescent stain in more than one sperm in the laser beam pattern. Each of these problems present in conventional flow cytometers reduces the ability to distinguish between events.
Another significant problem with conventional flow cytometry techniques, as well as others, is that irregularly shaped objects, such as sperm, produce different signals (shape, duration, or total number) depending on their orientation in the excitation/detection pathway. In this way, the individuals in a homogeneous population are able to produce a broad emission spectrum which is characterised by a possible overlap with the emission characteristics of the individuals in another homogeneous population, avoiding or reducing the ability to distinguish between the individuals in the two populations.
Another significant problem with conventional flow cytometer techniques, as well as others, is that the object is not uniformly exposed to the excitation source. Conventional beam shaping optics cannot provide uniform laser exposure when objects are close to the beam periphery.
Another significant problem with conventional flow cytometer techniques is that objects such as sperm are exposed to the excitation source for unnecessarily long periods of time. Irradiation of cells, such as sperm, with laser light may result in destruction of the cell or DNA within the cell.
Another significant problem with conventional flow cytometer techniques is that it can occur that the injection tube breaks the laminar flow membrane in the nozzle. Disruption of the laminar flow membrane changes the orientation of irregularly shaped objects in the fluid and reduces the speed of sorting and the purity of the sorted sperm population with the X-chromosome and the Y-chromosome.
Other problems may also exist with techniques that use stains to bind to the nuclear DNA of sperm cells. First, because the DNA in the nucleus is highly concentrated and flat in shape, stoichiometric staining of DNA is difficult or impossible. Second, the stained core has a higher refractive index. Third, the DNA-stain complex formed by the stain bound to the DNA may reduce the rate of insemination or the survival of the subsequent embryos. Fourth, the DNA-stain complex is typically irradiated with ultraviolet light to cause the stain to fluoresce. Such irradiation may affect the survival rate of the sperm. Because of these various problems, it would be preferable to use a method where the stain is less demanding or no stain is required, or where less or no ultraviolet radiation is required, or where both are less demanding or not required.
The present invention illustrates in a practical manner the solution of each of the above-mentioned problems for the generation of high purity samples bearing populations of X-chromosome bearing or Y-chromosome bearing sperm cells (whether viable, fixed, viable, non-viable, intact, tailless, or as nuclei), or, in general, for the detection of subtle differences in imaging signals between a series of events with relatively high incident light flux, or for the orientation of irregularly shaped objects in a fluid stream, or for the elimination of incidental events in an optical path, or for the removal of unwanted directional objects from analysis.
Disclosure of Invention
It is a significant object of the present invention to provide isolated populations of spermatozoa bearing highly pure X-chromosomes and Y-chromosomes. Isolated non-naturally occurring populations of high purity sperm cells have many uses, including sex selection of mammalian offspring, various in vitro fertilization protocols, various in vivo fertilization protocols such as artificial insemination, commercial methods including breeding rare or meat animals, or protecting rare or endangered animals, to name but a few of the uses of high purity sperm cell populations.
Another significant object of the invention relates to an apparatus and method for producing sperm samples bearing high purity X-chromosomes and Y-chromosomes.
One particular embodiment described herein may be used for the above-described multiple purposes, and may be used to successfully discriminate small measurable differences in the overall luminous flux of a light emission event, orient irregular objects in a fluid stream, minimize incidental events in an optical path, remove signals in an optical path contributed by unwanted unoriented objects (including removal of the objects themselves), and encapsulate irregularly shaped objects in a droplet. Thus, the specific objects of the present invention should be very broad.
It is another significant object of the invention to provide sperm samples (viable, fixed, viable, non-viable, intact, tailless, or sperm nucleus) having high purity X-chromosomes and Y-chromosomes with a gradient of high purity ranging from 80%, 85%, 90%, 95%, or even greater than 95%.
Another significant object of one embodiment of the invention can be to isolate populations of sperm cells bearing highly pure X-and Y-chromosomes, even at high isolation rates. The high speed separation to produce viable sperm of each sex may be at a rate of about 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or even 10000 or more per second.
It is another significant object of a particular embodiment of the invention to completely eliminate or remove sperm having an undesirable orientation (viable, fixed, viable, non-viable, intact, tailless, or sperm nucleus) in the excitation/detection portion of the flow path of a flow cytometer.
It is another important object of a particular embodiment of the present invention to provide an artificial insemination sample having a high purity of either the X-chromosome or the Y-chromosome.
It is another important object of a particular embodiment of the invention to provide in vitro fertilization samples having a high purity of either the X-chromosome or the Y-chromosome.
It is another important object of a particular embodiment of the present invention to enable preselection of the sex of offspring of females inseminated with high purity artificial insemination samples with a success rate of selection of offspring sex of insemination eggs inseminated with high purity artificial insemination samples of 80%, 85%, 90%, 95% or even more than 95%.
Another important object of a particular embodiment of the invention is to distinguish light emission events having small differences in total emitted light flux.
Another important object of particular embodiments of the present invention is to be able to completely eliminate or reduce the amount of background noise generated by a photomultiplier tube after exposure to high incident light flux for a period of time, even in the absence of light.
It is another important object of particular embodiments of the present invention to be able to completely eliminate the saturation of photomultiplier tube photocathodes used with flow cytometers or other instruments.
It is another important object of particular embodiments of the present invention to be able to reduce the number of electrons that migrate from the photocathode of the photomultiplier tube to the first dynode.
It is another important object of particular embodiments of the present invention to be able to reduce the total number of electrons flowing to the N-pole in a photomultiplier tube.
It is another important object of particular embodiments of the present invention to enable an increase in the luminous flux of a photomultiplier tube photocathode without a corresponding increase in the background signal produced by the photomultiplier tube.
It is another important object of particular embodiments of the present invention to be able to increase the ratio of the signal from the measured light emission event in the background signal.
It is another important object of particular embodiments of the present invention to enable an increase in the intensity of the amplified signal produced by a photomultiplier tube during a high incident light flux event or a succession of high incident light flux events without saturation of the cathode of the photomultiplier tube.
It is another important object of particular embodiments of the present invention to be able to improve the apparent resolution of chromatograms or histograms resulting from the classification of sperm or other cells or other objects stained with fluorescence, the excitation of which produces a slightly different luminous flux.
It is another important object of particular embodiments of the present invention to enable improved calibration of the device when sorting sperm using a sorting flow cytometer device.
It is another important object of particular embodiments of the present invention to be able to increase the sperm sorting rate of a flow cytometer system.
It is another important object of particular embodiments of the present invention to be able to improve the purity of sperm samples sorted by flow cytometry.
It is another important object of particular embodiments of the invention to provide a technique for separating X-chromosome bearing sperm from Y-chromosome bearing sperm that has a slightly different amount of Y-chromosome DNA and X-chromosome DNA from the total nuclear DNA count of the two sperm.
It is another important object of an embodiment of the present invention to provide a technique for increasing the apparent resolution of the resulting histograms during the step of separating X-chromosome bearing sperm from Y-chromosome bearing sperm using a flow cytometer.
It is another important object of particular embodiments of the present invention to be able to provide optical devices that shape the beam to minimize overlap of targets in the excitation/detection path.
It is another important object of particular embodiments of the present invention to provide an optical device that can provide a shaped beam that minimizes the total number of lumens of an object when exposed to an excitation beam. One aspect of this object is to reduce the overall brightness of the light intensity to which the object is exposed. A second aspect of this object is to increase the intensity of the light source without increasing the overall luminance of the exposed object.
It is another important object of particular embodiments of the present invention to provide beam shaping optics that uniformly expose an object as it passes through an optical path.
It is another important object of particular embodiments of the present invention to provide a nozzle that is capable of orienting irregular objects in a fluid stream. One aspect of this object is to make the objects directionally elongate in the same direction. A second aspect of this object is to orient the back side of the flat object in the same direction.
It is another important object of particular embodiments of the present invention to be able to completely encapsulate irregularly shaped objects within a droplet.
It is another important object of particular embodiments of the present invention to be able to distinguish objects in a fluid stream that do not require orientation from objects that require orientation.
It is another important object of particular embodiments of the present invention to provide a discriminatory interference contrast technique whereby a planar object is composed of a fluid stream with a target object and whereby the planar image can be used to measure signals from passing objects.
It is a further object of an embodiment of the present invention to be able to provide an optical arrangement to form images separating the two sides of each object, such that one is used to measure the actual volume of the object and the other is used to determine the orientation of the object. Thus, objects whose volume cannot be accurately measured because of incorrect orientation can be discarded. This can be done by modification so that the light pulses produced by these dual images are detected independently using two pinholes in the image plane. The optical device is tuned such that the light pulses produced by the first image are proportional to the volume of the object and the light pulses produced by the second image depend on the orientation of the object when measured.
It is a further object of an embodiment of the present invention to be able to provide a way of compensation in that the object is contained within the liquid flow. For example, the fluid stream may be a column of water that acts as a cylindrical lens, thereby distorting the image of the object. Optically, this corresponds to a cylinder having a higher refractive index (water) than its surroundings (air). Although other compensation elements of different shapes and refractive indices may be set as desired, the compensation disclosed in the present invention can be comprised of, for example, a cylinder having a lower refractive index than its surroundings. By ensuring that light passes through this compensation element, the optical effect of the liquid flow can be compensated by the exact opposite behaviour of the compensation element.
Naturally extended objects of the invention are disclosed throughout the remainder of the specification and claims.
Drawings
Fig. 1 shows a general flow cytometer.
Fig. 2 shows a second view of a typical flow cytometer.
FIG. 3 shows a comparison between univariate histograms (#1, #2, and #3) of the univariate flow cytometer of the invention without amplifier (FIG. 3a) -the univariate histogram of the embodiment of the invention utilizing a specific amplification (FIG. 3b), illustrating the improvement in resolution between the X-stained and Y-stained bovine sperm cell populations.
The univariate and bivariate histograms shown in FIG. 4 illustrate the conventional resolution between X-chromosome bearing and Y-chromosome bearing populations of bovine sperm cells.
FIG. 5 shows univariate and bivariate histograms illustrating the improved resolution between X-chromosome bearing and Y-chromosome bearing bovine sperm cell populations using a particular embodiment of the invention with amplification.
A second example of univariate and bivariate histograms shown in FIG. 6 illustrates the conventional resolution between X-chromosome bearing and Y-chromosome bearing bovine sperm cell populations.
FIG. 7 shows a second example of univariate and bivariate histograms illustrating the improved resolution between X-chromosome bearing and Y-chromosome bearing bovine sperm cell populations using a particular embodiment of the magnification invention.
The univariate and bivariate histograms shown in FIG. 8 illustrate the conventional resolution between X-chromosome bearing and Y-chromosome bearing equine sperm cell populations.
FIG. 9 shows univariate and bivariate histograms illustrating the improved resolution between X-chromosome bearing and Y-chromosome bearing equine sperm cell populations using a particular embodiment of the invention with amplification.
FIG. 10 shows univariate and bivariate histograms illustrating the improved resolution between X-chromosome bearing and Y-chromosome bearing equine sperm nuclei using a particular embodiment of the invention with amplification.
FIG. 11 shows a specific embodiment of an improved circuit board to which the present invention is applied in greater detailA flow cytometer.
FIG. 12 shows a view againstA circuit diagram of a particular embodiment of the invention is enlarged for a flow cytometer.
Fig. 13 shows the laser beam pattern of an optical device with a conventional shaped beam (fig. 13a) and the laser beam pattern of an optical device with a shaped beam of reduced height (fig. 13 b).
FIG. 14 shows a bar graph comparing the purity of X-chromosome bearing (FIG. 14a) and Y-chromosome bearing (FIG. 14b) sperm separated using conventional techniques either alone or in combination with the use of magnifying inventive techniques or optical means to reduce the height of the shaped beam.
Fig. 15 shows a front view of the beam shaping optics with reduced height.
Fig. 16 shows a top view of a beam shaping optics with reduced height.
Fig. 17 shows a perspective view and two intersecting sections of the object orienting nozzle invention.
FIG. 18 shows a series of stepped intersections of the object orienting nozzle invention.
FIG. 19 shows front and side views of an embodiment of the bevel injector invention.
FIG. 20 depicts the invention for the Removal of Unwanted Unoriented Sperm (RUUS) by comparing the signals for oriented sperm (FIGS. 20a and 20b) and for unoriented sperm (FIGS. 20c and 20 d).
FIG. 21 shows a perspective view of another embodiment of the bevel injector invention with a scored bevel blade.
FIG. 22 shows conventional optics in conjunction with a flow cytometer.
Fig. 23a shows typical sperm cell shape and size, while fig. 23b shows the difference between correctly and incorrectly oriented sperm cells.
FIG. 24 shows a configuration of an embodiment of the present invention capable of measuring both signals, e.g., volume and orientation.
Fig. 25a and b show a portion of an embodiment of the invention having pinholes corresponding to each semicircle, fig. 25c shows a plan view of an embodiment of the invention, and fig. 25d shows two independent rotatable polarizers of an embodiment of the invention.
FIGS. 26a and 26b illustrate a method of compensating for fluid flow according to an embodiment of the present invention, FIG. 26c shows a compensating element according to an embodiment, FIG. 26d shows another compensating element according to an embodiment, and the fluid flow and the image from the compensating element fall on top of each other in plan view.
FIG. 27 shows one embodiment of the interference optical device invention.
FIG. 28 shows a second view of the interference optics invention.
Detailed Description
The present invention relates to the isolation of sperm or populations of sperm cells bearing highly pure X-and Y-chromosomes. The population of high purity X-and Y-chromosome bearing sperm may include whole viable sperm, may include a population of tailless sperm (spermatozoa), or may take the form of other populations of viable or non-viable sperm, as desired. While each isolated whole viable sperm cell described in the context of the particular examples provided herein has the head, neck and tail of a sperm cell, it should be clear that the described techniques may also have a variety of different applications in the sperm nucleus. Populations of X-chromosome bearing and Y-chromosome bearing sperm should further be understood to include, but not be limited to, sperm from any male mammalian species, sperm from humans and from well known animals such as bovines, equines, ovines, canines, felines, caprines or porcines, as well as less commonly known animals such as elephants, zebras, camels, or striped antelopes. This list of animals is intended to illustrate the variety of animals whose sperm cells can be isolated by conventional methods at 90% purity or greater, and is not intended to limit the particular species from which sperm cells described in this specification can be derived from any mammal.
High purity sperm cells isolated from different species of mammals can be manufactured and used in various artificial insemination protocols or as part of a commercial process, such as described in those U.S. patent applications 60/211,093, 60/224,050 or PCT/US 99/17165; or may be used in a low dose insemination protocol as described in PCT/US98/27909 application, or for in vitro fertilization of oocytes of animals including humans as described in US patent application 60/253,785, each of which is incorporated herein by reference.
The term purity or high purity as used herein is to be understood as the percentage of isolated populations of spermatozoa having a particular distinguishing characteristic or combination of characteristics as desired. For example, where the population of spermatozoa is isolated on the basis of having an X-chromosome as opposed to a Y-chromosome, a population of X-chromosome bearing spermatozoa of 90% purity means that 90% of the individual spermatozoa in the population of spermatozoa present carry the X-chromosome and the other 10% of the population of spermatozoa may be Y-chromosome bearing spermatozoa. Thus, populations of spermatozoa bearing high purity X-chromosomes or Y-chromosomes should comprise a purity selected from the group consisting of between 90% and about 100%, between about 91% and about 100%, between about 92% and about 100%, between about 93% and about 100%, between about 94% and about 100%, between about 95% and about 100%, between about 96% and about 100%, between about 97% and about 100%, between about 98% and about 100%, and between about 99% and about 100%.
Importantly, while many of the embodiments of the present invention describe isolated populations of X-and Y-chromosomes bearing high purity sperm cells, and while the specification further discloses how the isolated populations of high purity sperm cells are isolated and utilized with high purity sperm cell isolation apparatus and methods, it should be clear that the basic concepts of the present invention can be applied to other types of particles or things having particle differentiating characteristics. It should be clear that the present invention can be applied to a wide variety of matters requiring the resolution of minute differences in imaging signals, such as product defect detection, field flow fractionation, liquid chromatography, electrophoresis, computed tomography, gamma imaging, time detection of flight testers, etc., which are easily understood by those skilled in the art.
Additionally, while this disclosure provides a description of embodiments of apparatus and methods for flow-separating X-chromosome bearing sperm cells and Y-chromosome bearing sperm cells, the description of these embodiments of the present invention is not intended to narrow the scope of the invention to flow separation of sperm cells only or to high purity flow cytometer sperm separation systems only, but rather these examples are used to illustrate the basic concepts of the present invention in a practical manner to illustrate their applicability to a wide range of applications.
Referring now to fig. 1 and 2, the flow cytometer of the illustrated embodiment of the invention includes a particle or cell source 1 that functions to immobilize or provide particles or cells stained with at least one fluorescent stain for analysis. These particles or cells are preserved in the nozzle 2 in such a way that they are introduced into the flow or sheath fluid 3. Some of the sheath fluid is usually supplied from the sheath fluid source 4 into the sheath fluid 3 so that when the particle or cell source 1 supplies the particles or cells to the sheath fluid 4, they are supplied in parallel through the nozzle 2.
From this form, it can be easily understood how the sheath fluid 3 forms a sheath fluid environment for the particle or cell. Since the various liquids are supplied to the flow cytometer at a certain pressure, they flow out of the nozzle 2 and out of the nozzle opening 5. By providing some type of oscillator 6 which can be accurately controlled by an oscillation controller 7, pressure waves can be established within the nozzle 2 and transmitted into the fluid exiting the nozzle 2 at the nozzle opening 5. As the oscillator 6 acts on the sheath fluid 3, the fluid 8 leaving the nozzle opening 5 eventually forms regularly arranged droplets 9. Because the particles or cells are surrounded by a fluid stream or sheath fluid environment, the droplets 9 may carry along within them individual, discrete particles or cells, which in certain embodiments of the invention are sperm cells.
Since the droplets 9 can carry away particles or cells, the flow cytometer may be used to separate particles, cells, or sperm cells and the like according to the characteristics of the particles or cells. This may be accomplished by a particle or cell sensing system 10. Such a particle or cell sensing system comprises at least some type of detector or sensor 11 that is responsive to the particles or cells contained in the fluid stream 8. The particle or cell sensing system 10 may respond based on whether it has a characteristic, such as a fluorescent stain incorporated into the particle or cell or DNA in the cell, that responds when excited by an illumination source such as a beam of light generated by the laser exciter 12 that causes the particle to respond. When the nuclear DNA of each type of particle, cell or sperm cell is stained with at least one fluorescent stain, the amount of fluorescent stain bound to each individual particle or cell will vary depending on the number of sites to which the particular type of fluorescent stain used is capable of binding. For sperm cells, the site to which Hoechst33342 stain can bind depends on the amount of DNA contained in each sperm cell. Since X-chromosome bearing sperm contain a greater amount of DNA than Y-chromosome bearing sperm, X-chromosome bearing sperm will bind a greater amount of fluorescent stain than Y-chromosome bearing sperm. Thus, by measuring the fluorescence emitted by the bound fluorescent stain after excitation, X-chromosome bearing sperm can be distinguished from Y-chromosome bearing sperm.
For separation purposes depending on the characteristics of the particles or cells, the emitted light may be received by the sensor 11 and fed back to some type of separation discrimination system or analyzer 13 connected to a droplet charger which charges each droplet 9 differently depending on the characteristics of the particles or cells contained in the droplet 9. In this way the separation discrimination system or analyser 13 acts to deflect the droplets 9 by the electrostatic deflection plates 14, depending on whether the droplets contain suitable particles or cells.
Thus, the flow cytometer functions to separate particles or cells 16 by directing the particles or cells to one or more collection containers 15. For example, when the analyzer distinguishes sperm cells based on their characteristics, a sperm droplet containing an X-chromosome is positively charged and thus shifted in one direction, while a sperm droplet containing a Y-chromosome is negatively charged in the opposite direction and thus shifted in the other direction, and the waste stream (droplets without particles or cells or with unwanted or unscreened cells) is left uncharged and thus collected in an aspiration tube or similar container in the non-shifted stream, as discussed in U.S. patent application 09/001,394, incorporated herein by reference. Naturally, many offset tracks can be established and collected simultaneously.
For routine separation of particles, cells, sperm cells or sperm (whole, viable, fixed, viable, non-viable or nuclear) into high purity X-chromosome bearing and Y-chromosome bearing populations, the particle identification device or method used should provide high resolution discriminatory features as the basis for analysis and separation.
With respect to sperm cells, as discussed above, it can be difficult to identify the difference between the light emitted by a fluorescent stain bound to the nuclear DNA of an X-chromosome bearing sperm cell and the light emitted by a fluorescent stain bound to the nuclear DNA of a Y-chromosome bearing sperm cell.
In many applications, the total emitted light from multiple emission events incident on a detector, which may be a photomultiplier tube, may be high, while the difference between the emitted light of the individual emission events to be distinguished may be small.
This problem is exacerbated when the light emission events occur continuously at a high rate and the time between light emission events is short, as is the case with high-speed cell screening using flow cytometry. When separating particles, cells or sperm cells according to the differentiation of the bound fluorescent stain, cells flowing past the excitation source can be generated and a large number of luminescence events occur per second. Thus, the number of particles, cells, or sperm cells in the fluid stream that can emit light can be substantial. As the flow rate increases, the intercept point of the excitation source becomes very bright. This high level of incident light on the photomultiplier photocathode results in a signal that is very low in proportion to the background signal. The amount of background signal is further exacerbated when the nuclear DNA of sperm cells is labeled with a fluorescent stain such as Hoechst 33342.
Many approaches to solving this problem have focused on reducing the total luminous flux of the photocathode tube by placing an optical filter in front of the photomultiplier tube. This method does not change the ratio of signal to background signal, and subsequent attempts to increase the sensitivity of the photomultiplier tube to light produce additional background signal, since the amount of signal from the background saturates the photomultiplier tube.
Typically, photomultiplier tubes operate at a voltage in the range of about 400 volts to about 900 volts. The lower linear operating limit of standard photomultiplier tubes such as photomultiplier tubes R928 and R1477 supplied by Hamamatsu corporation is about 300 volts. Thus, an apparatus or device equipped with photomultiplier tubes operates the photomultiplier tubes at 400 volts or more. Even if the number of anode electrons is reduced to the desired degree, as disclosed in U.S. Pat. Nos. 4,501,366 and 5,880,457, the voltage between the photocathode and the dynode one is kept high and reduction of the anode electrons is achieved by lowering the voltage of the remaining dynode or filtering out dark and shot noise inherent to the electrons.
Unexpectedly, reducing the voltage of the photomultiplier tube to below 400 volts to about 280 volts, or about 250 volts, or even to 0 volts, allows subtle differences in light emission to be distinguished even when the total amount of light emitted from each light emission event is high or even when the number of light train events occurring per second is large. The present invention allows the rate at which light emission events can be reached to increase to 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 during separation of X-chromosome bearing sperm and Y-chromosome bearing sperm for the rate of light emission events produced by irradiation of a fluorescent stain bound to the nuclear DNA of sperm cells, at least 20,000 separable events per second, at least 25,000 separable events per second, at least 30,000 separable events per second, at least 35,000 separable events per second, or higher.
As a specific example, an existing Cytomation SX is assembledFlow cytometers operate photomultiplier tubes at a low limit of 400 volts. The adjustable gain value operates the photomultiplier at a high voltage rather than a low voltage. SXThe flow cytometer may be modified by reassembling the photomultiplier tube modulator. The R16C resistor (2.49 kohm) of the third channel may be replaced with a 2.0K resistor to change the gain value of the amplifier to control the photomultiplier tube. This conversion allows the photomultiplier tube to be operated at about 280 volts. A similar switch may be an SX using two resistors with 3.75 kiloohms in parallelFlow cytometry, or using a 1.3 kilo-ohm resistor that allows the photomultiplier tube to be operated at a voltage of about 200 volts or slightly above zero volts, respectively. For this conversion, the medium density filter in front of the photocathode can also be eliminated, since the photomultiplier tube is operated outside the typical operating voltage range.
Unexpectedly, this conversion increases the signal to noise ratio of the light emission event due to the conversion of the signal into an electrical signal by the photomultiplier tube, and then by increasing the gain amplifier to the appropriate level of the analog to digital converter of the analyzer 13 to amplify the clearer signal and output the resulting univariate or bivariate histogram.
Referring now to FIG. 3, three different SXs were compared before using the present invention (FIG. 3a)(#1，#2，#3) univariate histograms of flow cytometers and shows the separation of intact viable ejaculated bovine sperm with the invention (FIG. 3 b). As can be appreciated from the univariate histograms, the degree of separation (apparent difference between X-chromosome bearing sperm and Y-chromosome bearing sperm represented by the peaks and valleys) of intact live X-chromosome bearing sperm 17 and live Y-chromosome bearing sperm 18 can be substantially improved with the present invention.
Using SX before utilizing embodiments of the inventionThe flow cytometer may have a separation or screening rate of about 17.9X10 for intact viable X-chromosome bearing sperm and Y-chromosome bearing sperm64.5 hours (i.e., in two streams-the first stream is X-chromosome bearing sperm and the second stream is Y-chromosome bearing sperm at a separation or screening rate of about 1,100 per second), the purity is about 87%, and the range is 84% to 93%. The rates for the three screened separable events were 22000, 23000, and 20000, respectively.
The average screening rate of viable sperm after the above transformation was about 40.3x106A purity of 90.8% for 4.5 hours (i.e., about 2,500 per stream per second), ranging from 89% to 92%. The event rates for the three screens were 13000, 15,000, and 19,500 per second, respectively.
It can be seen from the data that this embodiment of the invention not only results in an increase in purity of the isolated population of sperm but also results in a doubling of the rate of isolation or screening, with a substantial reduction in the rate of events that can be isolated.
Similarly, reference is now made to FIGS. 4 and 5, which illustrate the use of SX before use of the invention (FIG. 4) and after switching as described above (FIG. 5)Flow cytometer #1 bivariate histogram of complete live sperm screening of cattle. Prior to the use of the invention, 44Laser tuning to 135MW at 0 volts at photocathode, 1-fold gain and 1.0 medium density filter (1/10 amplitude) operation SXFlow cytometry, about 10,000 events per second. Next, SX was operated with the present invention with the photocathode at about 262 voltsFlow cytometer, laser adjusted to about 100MW, 4 x benefit and without medium density filter, about 10,000 separable events per second. As can be appreciated from these data, the increased peak-to-valley depth between the X-chromosome bearing population 19 and the Y-chromosome bearing population 20 demonstrates a substantial improvement in resolution.
Similarly, referring now to FIGS. 6 and 7, the use of SX prior to the use of embodiments of the present invention is shown under the same parameters as shown in FIGS. 3 and 4, respectivelyFlow cytometer #2 gave bivariate histograms (fig. 6) from the screening of bull whole live sperm and histograms (fig. 7) operated using an embodiment of the present invention. Again, a substantial improvement in resolution is demonstrated by the depth of the peak valley between the population 21 with the X-chromosome and the population 22 with the Y-chromosome.
Referring now to FIGS. 8 and 9, there is shown SX before (FIG. 8) and after (FIG. 9) use of an embodiment of the present inventionFlow cytometry is used to separate or screen whole live equine sperm to obtain bivariate histograms. When using embodiments of the present invention, live horse sperm are separated or screened using a photomultiplier tube at a voltage of less than 300 volts with a laser at 100mW power at a separation or screening rate in excess of 4,800 per second, averaging 12,000 events per second. The improved resolution of the X-chromosome bearing population 23 and the Y-chromosome bearing population 24 is surprising. Data displayIn contrast to the peak between 5 separation channels used in the present examples, about 8 to 9 separation channels were achieved using the present embodiments, and the purity of the population with the X-chromosome and the population with the Y-chromosome that had been screened was about 93%.
Referring now to FIG. 10, there is shown univariate and bivariate histograms plotted from the points of isolated equine sperm nuclei (S-05400) stained with Hoechst33342 screened using an embodiment of the present invention. The sperm nucleus is prepared from freshly ejaculated equine sperm. Sperm cells were washed by centrifugation, sonication and the resulting heads and tails were separated by Percoll density gradient centrifugation. The separated heads were fixed by washing with 2% formalin and then stained with Hoechst 33342. The stained nuclei were fixed with sodium azide (0.5%). Samples were run at 5000 events per second to make a histogram. The stained nuclei were then used to identify SX incorporating photomultiplier tubes transformed according to the above-described embodiments of the present inventionThe flow cytometer is calibrated. Compensation was used to calibrate bivariate renderings of both populations (stained X and stained Y). Note that both populations of equine sperm nuclei are shown as univariate plots, almost completely eliminated by baseline.
Referring now to FIG. 11, for SXA particular improvement in flow cytometry involves the use of two resistors in parallel to provide a correction value of 1.8K. The two 3.57K resistors 25 and 26 correspond to about 1.785K, which is close enough to the effective value. With this modification, the photomultiplier tube on this particular instrument can be operated at 200 volts. Naturally, other flow cytometers or other instruments using photomultiplier tubes may be modified to measure the amount of emitted light from a particular event. Fig. 12 provides an electronic overview of certain embodiments of the invention.
Another important embodiment of the invention is to reduce the height of the optical pattern of the beam. As shown in fig. 13a, the optical device forming the conventional illumination beam produces a beam pattern 27 that is taller or much taller than the sperm cell head 28 that passes through the beam. Thus, more than one sperm head containing a fluorescent stain bound to DNA enters the beam pattern at the same time. In this case, multiple sperm heads containing fluorescent stains bound to DNA may be simultaneously excited to fluoresce in a single emission event. Thus, the previous or subsequent emission events may include incidental light flux contributed by other sperm heads in the beam pattern 27. This results in a reduction in the difference in average light flux between light emission events that can discriminate between X-chromosome bearing sperm and Y-chromosome bearing sperm. This also results in a reduction in the difference in average light flux between the comparative light emission events for either X-chromosome bearing sperm or Y-chromosome bearing sperm. Importantly, the simultaneous excitation of fluorescent stains bound to multiple DNAs increases the average brightness of the events, such that the measurable luminous flux difference between events is a smaller percentage of the total emitted luminous flux. This makes it more difficult to quantify the differences between events.
By reducing the height of the beam shape, as shown in fig. 13b, the overlap of multiple sperm heads in the beam pattern 29 of reduced height during the same measurement event is reduced. This results in a greater average difference between light emission events that distinguish X-chromosome bearing sperm from Y-chromosome bearing sperm. This also enables the total average luminous flux measured for each emission event to be reduced. For a particular embodiment of the invention for screening bovine sperm having a height of about 9 μm, it was found that the height of the light beam may be about 20 μm. In the present application, it was found that a vertical beam height of less than 20 μm does not provide additional gain in resolution.
Referring to FIG. 14, it will be appreciated that the purity of the screened X-chromosome bearing population of bovine sperm (FIG. 14a) and the screened Y-chromosome bearing population of sperm (FIG. 14b) that have been stained with Hoechst33342 may be improved using an optical device that reduces the height of the beam pattern. This was true for both 25% and 40% of the univariate peaks of the screening channel. As can be further appreciated from FIG. 14, optical means that merely reduce the height of the beam pattern without relying on any other aspect of the invention may improve the purity of the separated sperm, such as the above described modifications to the photomultiplier tube circuitry (new PMT) in embodiments of the invention, or may be used in combination with the improved photomultiplier tube of embodiments of the invention to further improve the purity of the separated sperm sample.
Another advantage of the optical member that reduces the height of the beam pattern is that the time for the sperm to pass through the excitation laser beam or irradiate the beam can be reduced. Reducing the irradiation time of the excitation laser beam can reduce stress and damage to the sperm.
Referring again to fig. 14b, it will be appreciated that the reduced height beam pattern may be used in conjunction with an illumination beam pattern having a larger area than conventionally used. For example, a conventional elliptical beam pattern 27 as shown in FIG. 14a is about 30 μm 80 μm, whereas the beam used in the present invention to produce the best resolution between X-chromosome bearing sperm and Y-chromosome bearing sperm has a beam pattern 29 of 20 μm 160 μm for sorting bovine sperm. The area of the 20 μm 160 μm beam pattern is 1.3 times that of the 30 μm 80 μm beam pattern. In this case, the energy loss at the point of incidence is inversely proportional. This enables the intensity of the excitation laser to be increased without regard to damage of sperm by intensive irradiation. For example, if an optical device with a conventional beam pattern produces a 30 μm 80 μm radiation beam pattern and the energized laser is conventionally charged at 150mW, then the energized laser in a particular embodiment of the invention with a 20 μm 160 μm beam pattern is charged at 300mW without increasing the total intensity of the point of incidence. In addition, the excitation laser can be operated at 150mW to obtain the benefits of reduced irradiation energy per unit area, reduced irradiation damage, extended laser life, etc.
The invention of the optical means for reducing the height of the beam pattern and the invention of the photomultiplier tube amplification effect can improve the purity of the X-chromosome bearing and Y-chromosome bearing sperm population by about 4% or more as compared to conventional beam shaping optical means and conventional photomultiplier tube amplification devices.
The beam shaping optics 30 may be mounted to a flow cytometer as shown in fig. 15 and 16. As will be appreciated, light 31 emitted by laser excitation of a fluorescent stain bound to the DNA contained in the sperm can be detected by a photomultiplier tube 32 at 0 and 90 degrees relative to the sperm head plane 28 as it passes through the excitation laser beam pattern.
As can be appreciated, stained sperm must be aspirated in a precise pattern to pass through the excitation or illumination beam so that the head of each sperm is oriented with the surface plane of the sperm head in line with the photomultiplier tube zero degree detector. Accurate detection of sperm DNA content can only be determined by measuring the surface of the plasma-shaped sperm head 28. In this way, only those populations of sperm that can enter the excitation beam in the proper orientation can be accurately measured and classified according to DNA content.
Referring now to fig. 17, 18 and 19, certain embodiments of the invention may also be used to force the flat sperm head into proper orientation by a particle or sperm cell orienting nozzle 33 through hydraulic power as the sperm passes in front of the photomultiplier tube. As shown in fig. 17, the directional nozzle has an inner surface 34 that forms a similar conical shape. The internal taper changes from the circular shape of the inlet end 35 to the very elliptical shape of the outlet tip nozzle 36. The orifice 36 should be circular rather than elliptical. Thus, the internal aspect (internal aspect) of the directional nozzle 34 goes from a circular entrance to a narrow oval shape to a circular exit immediately in front of the orifice 36. The internal shape can be further elucidated by the intersection of the directional nozzles shown in fig. 18.
As shown in fig. 19 and 21, an injection tube 37 of about 0.061 inches in diameter may be used with a directional nozzle (or conventional nozzle) 33, which may be angled near the tip to form a blade 38. The flat vanes 38 are oriented at a 90 degree angle to the maximum radial direction of the ellipse in the directional nozzle 33. The injection needle may have an inside diameter of about 0.010 inches forming a circular nozzle 39 centered on the flat needle blade 38.
The paddle mounting of the angled blades is illustrated by FIG. 21 in a particular embodiment of the angled injection tube. The paddle-shaped inclined blades may help to maintain laminar sheath fluid flow in the nozzle (either a conventional nozzle or a directional nozzle). In this way, the laminar flow of liquid maintained by the paddle-shaped inclined blades shows less disruption to the substance injected therein. Sperm introduced into the laminar flow of sheath fluid is retained by the injector tube with paddle-shaped angled vanes in a particular embodiment of the invention and the sperm sorting rate is increased by 20%, 30%, 40%, 50% or even more than with conventional injector tube technology. High sperm sorting rates may achieve sorting rates of about 4,000 to 10,000 per second per sex. High purity (90% or even higher) populations of X-bearing chromosomes and Y-bearing chromosomes can be produced even at such high sorting rates. The invention of a syringe barrel with an angled paddle tip may be used alone or in combination with other inventions described herein, such as other techniques described in U.S. patent application 09/454,488 or PCT/US00/42350, which are incorporated herein by reference.
As shown in FIG. 21, embodiments of the inclined blade injection tube invention or the inclined paddle blade invention may further include laminar flow enhancing grooves 40. The laminar flow enhancing groove 40 helps to maintain laminar flow into the nozzle of the syringe barrel. Again, the enhanced laminar flow keeps more sperm correctly oriented in the sheath fluid resulting in a higher sortable event rate and subsequently an increased sorting rate for each sex or sperm.
In another embodiment of the invention, the directional nozzle opening 39 or other conventional nozzle opening size can be adjusted so that the formed droplets encapsulate intact viable sperm exiting the nozzle opening 39. There is no technique for encapsulating sperm cells in conventional sperm cell entrainment techniques. A significant portion of the sperm cell tails remain outside the droplet. For example, bovine sperm cells have a length of about 13.5 microseconds when the pressure of the fluid stream is about 50 pounds per square inch (i.e., the length of time that a full length sperm cell passes through the illuminating light beam at a fluid stream pressure of 50 pounds per square inch). Conventional drop formation techniques for entraining bovine sperm cells form a 14 microsecond drop (i.e., the time required to form a waveform of drops in a stream) from a nozzle having an orifice of about 70 microns in diameter, which can respond to an oscillator operating at 35 kilohertz. Thus, a portion of the sperm cell tail tends to protrude from the droplet. To prevent the sperm cell tails from protruding from the droplet, one embodiment of the droplet encapsulation invention provides an orifice of about 100 microns that produces a droplet of about 28 microseconds at about 30 kilohertz at about 50 pounds per square inch. By complete encapsulation of intact viable sperm cells, including the tails, the interaction between sperm cells and the nozzle due to droplet charging is less and the deflection of the droplets is more precise. This results in reduced cross-contamination between X-chromosome bearing sperm and Y-chromosome bearing sperm and also allows for more uniform collection of offset sperm. The consistently deflected sperm are directed to a collection surface that is buffered by different liquids. Buffering of separated sperm cells is important to reduce stress, as described in U.S. patent application 09/001,394, which is incorporated herein by reference. The invention can be modified to produce droplets of various sizes to encapsulate sperm cells of different lengths for sperm from other mammalian species. Depending on the length of the sperm and the pressure of the stream, the droplet encapsulation invention can still achieve a droplet formation rate 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 until reaching about 200,000 droplets per second in some embodiments of the droplet encapsulation invention.
Even with the directional nozzle invention, there is still a certain amount of incorrectly oriented sperm or particles in the beam pattern. As described above, if the orientation of the sperm head is incorrect, the DNA content measured from the emitted light is inaccurate. Particular embodiments of the present invention provide for the removal of unwanted incorrectly oriented sperm (RUUS) or particles in a fluid stream.
Referring now to fig. 16 and 20a, it can be appreciated that accurate determination of sperm DNA content depends on the orientation of the paddle-shaped sperm head 28 surface to the detector being correct. Thus, only those portions of sperm that entered the excitation beam in the correct orientation as shown in FIGS. 16 and 20a can be accurately determined and separated into X-chromosome bearing and Y-chromosome bearing populations based on DNA content. As shown in FIGS. 20a and 20b, sperm passing the excitation beam in the correct orientation produce a directed emission signature 40 that is shaped differently than the undirected emission signature 41 produced by an undirected sperm as shown in FIG. 20 d. Naturally, the variation in the pattern 41 of unoriented emission signals produced by unoriented sperm is dependent on the degree of incorrect orientation of the sperm in the excitation beam. These incorrect orientations include the orientation shown in fig. 20c, but also include all forms of turning the orientation of the sperm head, either a partial rotation such that the orientation of the paddle head surface is not aligned with the detector (correct alignment as shown in fig. 16), or any rotation such that the orientation of the axis 42 of the sperm head is turned out of alignment with the flow stream. Naturally, the definition of the correct orientation between different species is different. For some species in which the sperm head is not paddle-shaped, the correct orientation of the sperm in the excitation beam, or relative to the detector, or vice versa, may be defined by other anatomical or signal characteristics. However, the optimal signal generated by sperm of the different species oriented correctly within the excitation window can be used as a standard emission signal profile for a series of luminescence events to be compared later.
By comparing the shape of each emission signature (or the entire region or both) with the standard emission signature determined for oriented mammalian sperm (or the standard entire region or both), unoriented sperm can be identified, subtracted from the univariate or bivariate histograms, and if desired, definitively removed so that unoriented sperm are neither collected in the X-chromosome bearing population nor in the Y-chromosome bearing population.
Importantly, the present invention increases the resolution between two populations of spermatozoa to be separated thereby increasing the rate at which one population is separated from the other and increasing the purity of the separated populations. Thus, sperm can now be sorted at extremely high speed. The rate of classifiable or separable events can reach about 35,000 events per second (excluding coincident events-multiple sperm are simultaneously in the excitation/detection window). The high separation or sorting rate, which correlates to the rate of sortable or separable events, can be from about 5000 to about 11,000 whole viable sperm of each sex per second and have a purity of 90%, 92%, 93%, 95% or more. The invention described above can enable even higher purity X-chromosome bearing and Y-chromosome bearing populations to be obtained at about 97% to about 98% or even higher purity by reducing the sorting or separation rate to about 2000 viable sperm of each sex per second.
As can be appreciated, the above-described invention is particularly important for achieving the highest possible rate of sorting or separation events and the highest possible separation rate that can be achieved, which can be at least 1,000 isolates per species per second, at least 2,000 isolates per second, at least 3,000 isolates per second, at least 4,000 isolates per second, at least 5,000 isolates per second, at least 6,000 isolates per second, at least 7,000 isolates per second, at least 8,000 isolates per second, at least 9,000 isolates per second, at least 10,000 isolates per second, or higher.
As described above, the present invention can provide for high speed sorting of sperm cells, even when staining of such sperm cells is difficult, or where differentiation between X-chromosome bearing and Y-chromosome bearing populations is more difficult due to other anatomical or chemical properties. Even in these difficult situations, it is still possible to achieve separation of high purity X-chromosome bearing and Y-chromosome bearing populations of bovine sperm at 92% to 93% purity and sorting or separating intact viable sperm of each sex (X-chromosome bearing and Y-chromosome bearing) at a rate of 2000 intact viable sperm of each sex per second by providing a rate of separable events of about 15,000 and 20,000 separable events per second as described above.
Referring now to fig. 23 and 24, one embodiment of the present invention utilizes a differential interference contrast technique to measure the volume of a particle or capsule. A basic embodiment of the invention may include a head 28 of sperm cells having different volumes, such as X-chromosome bearing and Y-chromosome bearing sperm cells. The electromagnetic radiation source 43 produces a beam 44 of electromagnetic radiation or electromagnetic radiation having an initial waveform characteristic that produces a different response to the volume difference between the particle or sperm cell heads 28. The electromagnetic radiation may be laser light, but may also be various types of electromagnetic radiation, including but not limited to microwave radiation, ultraviolet radiation, and the like. When passing through the volume of the particle or capsule or sperm head containing the phase shifting material, the electromagnetic radiation is focused by an objective lens 45 onto a detector 46 responsive to the electromagnetic radiation waveform characteristics. The detector may be used in conjunction with the analyzer 47. The analyzer can distinguish particles from changes in waveform characteristics before and after passing through the particle volume and can analyze the signal as a function of the integrated area or the shape of the signal or both. In certain embodiments of the invention, analyzing the waveform characteristics comprises superimposing an initial waveform characteristic with a modified waveform characteristic as it traverses the volume of the particle, capsule or sperm cell head. Superimposing the initial waveform characteristic and the phase-shifted waveform characteristic enables the intensity of the beam of electromagnetic radiation to be individually adjusted in relation to the amount of phase-shifting medium traversed by the electromagnetic radiation. The present invention should also include an additional filtering device 48 such as a colored filter.
Referring now to fig. 24, embodiments of the optical device invention involve the use of differential interference contrast optics that enable actual distances to exceed optical fractures as compared to conventional DIC microscopy consistent with the limits of microscope resolution. In this embodiment of the invention, the induced cracks are larger than the size of the object, thus resulting in two separate images originating from one object that are laterally separated. A second improvement involves the use of a plate of birefringent material, such as a Savart plate, at a position away from the objective lens. Embodiments of the present invention are easy to construct since the birefringent material need not be placed in the objective lens housing. The birefringent material used in conventional DIC microscopes is a prism known as Wollaston, which has to be placed in an objective box, which has to use expensive objective lenses specially produced for this purpose.
The components of the present embodiment may be aligned with each other and may be formed from an electromagnetic radiation source 43, such as a mercury arc lamp; spectral tuning components, such as band pass filters; polarization modifying components 49, such as a thin layer polarizer 53 and a lightwave plate 54 responsive to a rotatable slide; a condenser 51, such as a condenser lens, or set of lenses, or microscope objective, capable of focusing light onto the particles or sperm cells; a liquid stream 8 containing particles or sperm cells 28, such as liquid ejected from a nozzle under pressure; a light collector 45 to collect light from the particles or cells, such as a 50 x high working distance microscope objective and tube lens; a beam splitting device 50, for example a sheet of birefringent material in the form of a Savart plate, which splits the beam into two or more components, is mounted in a form in which the orientation and position can be precisely controlled; an image optical selector that selects only the light corresponding to the particle or sperm cell, for example a series of pinholes, each pinhole 53 being composed for each image formed.
In one embodiment of the invention, the components may be mounted in a form commonly referred to as Kohler illumination, i.e. the light source 43 or its image is located on the back focal plane of the condenser 45. The planar image of the object preferably coincides with the object's light selector 55 or pinhole 53 to capture light from individual particles or sperm. The components may be mounted on a stable optical table or table using fittings, braces, and brackets as shown in fig. 27 and 28. The components may be mounted in some form so that the object plane can be accurately focused. This may be accomplished by loading the stream with a stream position controller, such as a micrometer, to turn the stream into or out of focus. The condenser 51 may additionally be equipped with a condenser position control 61 to focus it on the object plane. Particular attention is paid to the assembly of the birefringent member or beam splitter 50, preferably a three-axis rotating member.
Referring now to fig. 25, embodiments of the invention may also include using two generated images to determine the orientation of asymmetric particles in a fluid stream, including but not limited to sperm such as bovine sperm cells. Embodiments of the orientation determination of the present invention include optical means to facilitate control of the polarization state of light entering a system that enables two independent images to be generated. The invention of the interference optics may further provide a polarization modifying element 56 that controls the polarization state of light entering the device. For the orientation detection invention, the selection of the polarization modifying element 56 may be made of two parts that are imaged on the image optical selector 55 with the pinhole 53 in one embodiment of the invention. This can be done by placing the polarization modifying component 56 in the middle of the junction plane of the image plane 55, or using other optical means to do the same. A simple example of such a component is a 'penumbra' sheet, for example consisting of two semi-circular sections of polarised material, such as sheet polarisers of which the orientation angle can be independently selected. Each pinhole in the image plane may fall into one of the hemispheres. The polarization angle may be chosen such that one pinhole signal 53 corresponds to the volume and is relatively independent of the orientation angle of the passing object, while the other pinholes have signals 53 that depend to a large extent on this orientation angle. Both signals may be processed by the analyzer 47 in a manner similar to that of a conventional multi-channel flow cytometer, but as an example only. For this embodiment, a double variable dot pattern can be made, and the user is allowed to select holes on this pattern as well.
A modification to the 'penumbra' sheet as described above may be a configuration as shown in figure 25 d. The two identical hemispherical portions are projected onto the image plane but are produced in different ways. The mirror 57 splits the light 44 into a semi-circle and recombines them back-to-back. Each half circle passes through a separate tool that controls its polarization state. The advantage of this embodiment is that the polarization angle can be controlled continuously and independently, thus facilitating adjustment of the device. The materials used in this embodiment can be supplied by standard optical suppliers and can be installed in equipment using similar assembly materials used for interference optics.
Referring now to FIG. 26, to correct for artifacts introduced by passing light through non-planar regions of a transparent substance, such as a generally cylindrical flow but also including other geometric shapes, embodiments of the present invention disclose a bonding element that is similar in shape to the non-planar regions but has an opposite relative refractive index. A particular case in flow cytometry is an approximately cylindrical shape. The correction of the artificial artefacts introduced by the fact that the object to be evaluated is placed inside the columnar flow of water is carried out by incorporating an optical element 58 capable of forming a transparent cylindrical shape in a transparent material 59 of higher refractive index. It is preferred that the images of the fluid stream and the compensating element lie on top of each other in the respective image planes. This can be done by placing a compensation element between the objective lens and the image plane in combination with the use of an auxiliary lens.
One embodiment of the optical component 58 is placed in a sheet 59 of a transparent material with a higher refractive index, such as glass or plexiglass, with a cylindrical hole drilled in the material. The advantage of plexiglass is that it is relatively easy to drill a circular channel into it. The cylindrical holes may be filled with a transparent material having a lower refractive index than the surrounding material. The difference in refractive index between the material and its surrounding material is the same as, but opposite to, the difference in refractive index between water in the stream and the surrounding air in some applications. It is not necessary to make the cylinder the same size as the water stream, as long as the magnification of the lens used is such that the image obtained in the image plane is of the same size. In some applications, it is desirable or necessary to adjust the difference in refractive index to compensate for this magnification. The manufacture of such elements from plexiglas is very simple and can be done by numerous mechanical workshops that have been experienced with mechanical tooling of the plexiglas or of the chosen material. The specifications may be prepared to meet the standards for optical assembly parts for incorporation into optical devices.
A perfectly matched refractive index is difficult. One convenient embodiment of the present invention is to have the organic glass, or other material of choice-a transparent refractive index liquid 58-be an organic oil, or a mixture of oils having a similar refractive index to the desired one, but these are examples only. Since many liquids have refractive indices that vary more readily with temperature than do solids or glasses, it is possible to fine tune the difference in refractive index by temperature. This may be accomplished by connecting a temperature controller 60.
The transparent liquid or refractive index liquid for optical component 58 may be supplied by a chemical supplier. These manufacturers typically have data on the refractive indices of the liquids they are available for. Some manufacturers even provide liquids that are specific for refractive liquids and have a guaranteed and stable refractive index. Temperature controllers and thermostats are provided by many manufacturers. One practical way of applying heat to the refractive index liquid is to prepare a hollow device, containing the refractive index liquid, from a thermally conductive material, such as a metal (by way of example only). With the conventional immersion type thermostatic circulation control device owned by many laboratories, water can be pumped through the device to keep the temperature of the element stable and controllable.
The discussion in this PCT application is intended to serve as a basic description. The reader should be aware that the specific discussion does not describe in detail all possible embodiments; many alternatives are not described. Nor does it fully explain the generic nature of the invention and 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 implicit in the present disclosure. Where the invention is described in terms of functionality, each aspect of functionality is implemented by a device, subroutine, or program. The apparatus claims should not be included solely in the described apparatus, but rather should be included to describe the method or steps for describing the invention and the function performed by each element. Neither the description nor the terminology is intended to limit the scope of what is not covered by the claims.
In addition, each of the various elements of the invention and claims may be accomplished in a variety of ways. It is to be understood that the present disclosure includes each such variation, and can be a variation of any apparatus embodiment, a variation of a method or step embodiment, or even merely a variation of any element thereof. In particular, it should be understood that as the disclosure relates to elements of the invention, the words used 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 specification of each element or action. Such terms may be substituted where necessary to elaborate the broad scope covered by the claims of the present invention. As but one example, it should be understood that all actions may be expressed as a means for performing that action or as an element which results in that action. Similarly, it should be understood that each of the physical elements disclosed includes the function facilitated by that physical element. With respect to this last aspect, as but one example, it should be understood that disclosure of "droplet separator" includes disclosure of the "separating droplets" effect-whether or not discussed in detail-and vice versa, if only the "converting liquid-gas" effect is disclosed, it should be understood that such disclosure includes disclosure of "droplet separator" and even of the "method of separating droplets". It is to be understood that such alternatives and modifications are expressly included in the present description.
In addition, various combinations and modifications of all elements or applications may be established and occur. All of which may be used to optimize the design or performance of a particular application.
Any law, act, regulation, or act of regulation mentioned in this application to obtain patent rights: patents, or publications, or other reference materials referred to herein for patenting are hereby incorporated by reference. Specifically, U.S. patent applications 60/267571, 60/239,752, and 60/203,089 are all incorporated by reference herein, including any data or attachments, and each reference in the following tables is incorporated by reference.
| Publication number | Date | Name (R) | Class I | Subclass of | Date of filling |
| 32,350 | 02/10/87 | Bhattacharya | 204 | 180.1 | 11/22/74 |
| 3,687,806 | 08/29/72 | Van den Bovenkamp | 195 | 1.3 | 11/04/69 |
| 3,829,216 | 08/13/74 | Persidsky | 356 | 36 | 10/02/72 |
| 3,894,529 | 07/15/75 | Shrimpton | 128 | 1R | 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 | Bhattacharya | 204 | 180R | 10/20/76 |
| 4,155,831 | 05/22/79 | Bhattacharya | 207 | 299R | 02/23/78 |
| 4,191,749 | 03/04/80 | Bryant | 424 | 105 | 10/11/77 |
| 4,225,405 | 09/30/80 | Lawson | 204 | 180R | 08/16/78 |
| 4,276,139 | 06/30/81 | Lawson | 204 | 180R | 10/09/79 |
| 4,339,434 | 07/13/82 | Ericcson | 424 | 561 | 08/17/81 |
| 4,362,246 | 12/07/82 | Adair | 209 | 3.3 | 07/14/80 |
| 4,448,767 | 05/15/84 | Bryant | 424 | 85 | 02/15/80 |
| 4,474,875 | 10/02/84 | Shrimpton | 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 |
| 4,660,971 | 04/28/87 | Sage et al | 356 | 39 | 05/03/84 |
| 4,680,258 | 07/14/87 | Hammerling et al | 435 | 7 | 08/09/83 |
| 4,698,142 | 10/06/87 | Muroi et al | 204 | 182.3 | 07/31/85 |
| 4,749,458 | 06/07/88 | Muroi et al | 204 | 182.3 | 03/02/87 |
| 4,988,619 | 01/29/91 | Pinkel | 435 | 30 | 11/30/87 |
| 4,999,283 | 03/12/91 | Zavos et al | 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 et al | 435 | 2 | 04/25/94 |
| 5,483,469 | 01/09/96 | Van den Engh et al | 364 | 555 | 08/02/93 |
| 5,503,994 | 04/02/96 | Shear, etc | 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 et al | 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 | Rens et al | 422 | 073 | 07/24/97 |
| 6,071,689 | 06/06/00 | Seidel et al | 435 | 2 | 01/29/98 |
| WO96/12171 | 13/10/95 | ||||
| WO98/34094 | 06/08/98 | ||||
| WO99/05504 | 07/24/98 | ||||
| WO99/33956 | 08/07/99 | ||||
| WO99/38883 | 05/08/99 | ||||
| WO99/42810 | 26/08/99 | ||||
| WO00/06193 | 10/02/00 |
In addition, it should be understood that each term used, unless otherwise interpreted as having a different meaning as applied to the present application, is intended to be interpreted as including every term and all definitions, alternative terms, and synonyms such as those set forth in the second edition of the blue-book-house weber dictionary, which is incorporated by reference herein. However, to the extent that such information or statements incorporated by reference herein can be extended in each of the above respects, it is not to be considered as inconsistent with the authorization of this/these inventions, and such statements are expressly not to be considered as made by the applicants.
Further, unless the context requires otherwise, it should be understood that the term "comprises/comprising" or any other verb form is intended to specify the presence of stated elements or steps or a group of elements or steps but does not exclude the presence of any other elements or steps or a group of elements or steps. Such terms should be construed in their broadest form to confer the applicant the broadest legal permission in australia and the like.
It is, therefore, to be understood that at least in part I) each of the liquid-gas shift devices described herein, ii) the related methods disclosed and described, iii) similar, equivalent, and such apparatus and methods as may actually imply changes, iv) alternative designs and methods as disclosed and described that are capable of performing each of the functions shown, v) those alternative designs and methods as may be capable of performing or are meant to be capable of performing each of the functions shown as described and disclosed above, vi) the features, components and steps of the separate and independent invention shown, vii) applications for improvement by the different systems or components disclosed, viii) products derived from such systems or components, ix) methods and apparatus substantially as described herein above and with respect to any additional embodiments, and x) various combinations and permutations of each and every disclosed element.
Further, unless the context requires otherwise, it should be understood that the term "comprises" or other forms of verbs are intended to be inclusive of the stated element or step or group of elements or steps but not to exclude any other element or step or group of elements or steps. Such terms should be construed in their broadest form to confer the applicant the broadest legal permission in australia and the like.
The claims set forth in this specification are hereby incorporated by reference into the specification as if fully set forth herein, and the applicants expressly reserve the right to incorporate some or all of these claims as an add-on to support all or part of the claims, or any part or element thereof, and the applicants further expressly reserve the right to move some or all of these claims, or any part or element thereof, incorporated herein by reference from the specification to the claims, or vice versa, as if the application or any subsequent application, divisional application, or portion thereof continues to claim, to define what is claimed, or to obtain any benefit from the reduction of such claim, as per the provisions of the patent laws, rules, or treaties in any country, which are incorporated herein by reference, including any subsequent application, the divisional application, or a portion of a subsequent application, or the entire period over which modifications or extensions are made, should be effective.
Claims (12)
1. A flow cytometer, the flow cytometer comprising:
a radiation source capable of generating a radiation beam responsive to a sperm cell having a sperm cell distinguishing characteristic in a fluid stream;
an optical device that focuses the radiation beam;
a detector responsive to light emitted from a light-emitting material associated with each sperm cell in the fluid stream, wherein the light-emitting material produces emitted light in response to the radiation beam in accordance with the sperm cell distinguishing characteristic; wherein the sperm cell distinguishing characteristic comprises a sperm cell orientation characteristic of the sperm cell within the fluid stream, and wherein the detector produces a different response to the light emitted from the light emitting material based on the sperm cell orientation characteristic; the light emitting material associated with the sperm cell comprises a fluorescent stain associated with the nuclear DNA of the sperm cell; the sperm cell differentiating characteristic further comprises a difference in the amount of fluorescent stain bound to the nuclear DNA of the X-chromosome bearing sperm cell and the Y-chromosome bearing sperm cell;
an analyzer connected to the detector, the analyzer differentiating the sperm cells according to their orientation in the fluid stream, and wherein
The optical device focuses the radiation beam to form a radiation beam pattern having a height equal to the length of the sperm cell along its longitudinal axis to three times the length of the sperm cell along its longitudinal axis.
2. The flow cytometer of claim 1, wherein at least one of the sperm cells has a head with a length along a longitudinal axis of 9 microns, and wherein the radiation beam pattern has a height of 20 microns.
3. The flow cytometer of claim 1, wherein the detector comprises at least one photomultiplier tube having a typical operating voltage range of 400 volts to 999 volts, and the photomultiplier tube is operated in a range from 0 volts to 300 volts.
4. The flow cytometer of claim 1, wherein the flow cytometer further comprises:
an oscillator adapted to operate at 30 khz, the oscillator being connected to a directional nozzle and adapted to create a pressure wave within the directional nozzle, the pressure wave being capable of being transmitted to a sheath fluid surrounding the sperm cells, which together exit the directional nozzle to eventually form droplets;
a droplet charger coupled to the analyzer, wherein the droplets are charged differently based on a difference in the amount of stain bound to nuclear DNA of X-chromosome bearing sperm cells and nuclear DNA of Y-chromosome bearing sperm cells;
a pair of electrostatic deflection plates for deflecting the charged droplets; and
one or more collection vessels to collect the charged droplets.
5. The flow cytometer of claim 4, wherein the droplet formation has a droplet formation rate selected from the group consisting of 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.
6. The cytometer of claim 4 wherein the orientation nozzle has an internal aspect from a circular entrance to a narrow oval to a circular exit and an internal surface that forms a cone-like shape that in combination forces the flat sperm head to orient correctly by hydrodynamic forces as the sperm pass in front of the photomultiplier tube.
7. The flow cytometer of claim 6, wherein the directional nozzle has a nozzle orifice of 100 microns diameter.
8. The flow cytometer of claim 1, wherein the flow cytometer further comprises an injection tube that can be tilted near a tip to form a flat blade.
9. The flow cytometer of claim 6, wherein the flow cytometer further comprises an injection tube that can be tilted near the tip to form a flat blade, and wherein the flat blade is oriented at an angle of 90 degrees from the maximum radial of the ellipse in the orientation nozzle.
10. The flow cytometer of claim 8, wherein the flat blade is paddle-shaped.
11. The flow cytometer of claim 10, wherein the injection tube further comprises a laminar flow enhancement groove.
12. The flow cytometer of any of claims 1,3, 4 or 8 wherein the optical means is capable of providing a beam pattern for optimal resolution between X-chromosome bearing sperm and Y-chromosome bearing sperm for sorting bovine sperm having a beam pattern of 20 μm X160 μm.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US20308900P | 2000-05-09 | 2000-05-09 | |
| US60/203,089 | 2000-05-09 | ||
| US23975200P | 2000-10-12 | 2000-10-12 | |
| US60/239,752 | 2000-10-12 | ||
| US26757101P | 2001-02-10 | 2001-02-10 | |
| US60/267,571 | 2001-02-10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1163810A1 HK1163810A1 (en) | 2012-09-14 |
| HK1163810B true HK1163810B (en) | 2014-10-03 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5071995B2 (en) | High purity sperm population with X and Y chromosomes | |
| US4765737A (en) | Cell size measurements using light in flow cytometry and cell sorting | |
| CN102119212B (en) | Method and apparatus for sorting cells | |
| EP0229815B1 (en) | A device for measuring the light scattering of biological cells in flow cytophotometers | |
| US6813017B1 (en) | Apparatus and method employing incoherent light emitting semiconductor devices as particle detection light sources in a flow cytometer | |
| Parks et al. | Flow cytometry and fluorescence-activated cell sorting | |
| JPS63118638A (en) | Method and device for determining characteristic of cell, particle, etc. by using multiple fluorometric analysis | |
| US20240042491A1 (en) | Systems and methods for particle sorting with automated adjustment of operational parameters | |
| CN111044435B (en) | Multi-index detection light path system for flow cytometer | |
| HK1163810B (en) | A flow cytometer | |
| HK1201552B (en) | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa | |
| HK1151433A (en) | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa | |
| HK1151432A (en) | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa | |
| HK1151691A (en) | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa | |
| HK1151434A (en) | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa | |
| Steinkamp | Identification of Single Cells by Fluorescent and Darkfield Optical Techniques | |
| SCHROEDER et al. | Introduction to Flow Cytometry | |
| HK1151434B (en) | High purity x-chromosome bearing and y-chromosome bearing populations of spermatozoa |