CN103013811A - Sperm sorter - Google Patents
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- CN103013811A CN103013811A CN2012103520511A CN201210352051A CN103013811A CN 103013811 A CN103013811 A CN 103013811A CN 2012103520511 A CN2012103520511 A CN 2012103520511A CN 201210352051 A CN201210352051 A CN 201210352051A CN 103013811 A CN103013811 A CN 103013811A
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- sorter
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- excitation device
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
Abstract
The invention discloses a sperm sorter, relating to the technical field of cell biology. The sorter comprises a nozzle, wherein the lower end of the nozzle is connected with a spray needle, the needle head of the spray needle is wedge-shaped, and the inner portion of the nozzle is in the shape of a flat oval. The sperm sorter disclosed by the invention is formed by carrying out improvement on the basis of the traditional flow cytometry, so that the efficiency and accuracy of sperm separation are greatly improved, and the separation speed is increased by nearly 3-5 times.
Description
Technical field
The present invention relates to the cellular biological technique field, particularly a kind of sperm sorter.
Background technology
Existing traditional flow cytometer, such as grant number be: CN98813255.9, name is called: the patent that Mammals is had the insemination of sex selection with a small amount of spermoblast, it discloses a kind of to the improved drain cell calculating instrument of the required cell system of classifying, this System Working Principle is: sample to be measured is prepared into single cell suspension and enters the flow chamber that is full of sheath fluid after by fluorescent dye, the sheath hydraulic coupling is different from sample flow pressure, when the pressure gap of the two acquired a certain degree, sheath fluid was sweeping along that the cell defiled passes through the laser focusing district one by one in the sample flow.If we are with the specific fluorescence dye of putting on of interested part in the cell, so these dyestuffs will cell during by the laser detection district Stimulated Light send the fluorescence of specific wavelength, colour filter by certain wavelength selective permeability, we can make a distinction scattered light, the fluorescent signal of different wave length, and deliver in the different photomultiplier, through a series of conversion of signals, amplification, digitized processing, we just can add up the cell percentage separately of catching various fluorescence dyes intuitively at computer.Select different mono-clonal, clonal antibody and fluorescence dye, we can measure the multiple different characteristics on the cell simultaneously; If interesting to the cell with certain feature, we can also utilize the sorting function of streaming that it is sorted out, in order to further cultivate, study.
In Mammals, determine that the key of Animal Sex is its entrained different sex chromosome.For example normal amphiploid ox always has 60 karyomit(e)s, and wherein bull is 58 euchromosomes, 1 X chromosome, 1 Y chromosome; And cow is 58 euchromosomes, 2 X chromosomes.When through after reduction division forms haploid gamete, the x sperm carries X chromosome, produces female offspring behind it and the ovum fertilization; Y sperm carries Y chromosome, produces male offspring behind it and the ovum fertilization.In Mammals (domestic animal), the dna content of x sperm Duos 3.0 ~ 4.5 than the dna content of y sperm usually, can utilize fluorescent dye that this difference is distinguished by traditional flow cytometer, thereby reach the purpose of screening.
The tradition flow cytometer adopts dye Hoechst 33342 to dye when separated sperm, usually first sperm is diluted to 1.5 hundred million/milliliter, adds Hoechst33342 to 40 ug/ml, and dyeing is 60 minutes in 35 ℃ of water-baths.When sperm was by flow cytometer after the dyeing, defiled in trickle sample feeding pipe liquid stream was handed over laser one by one behind the high speed injection nozzle and is cut.Dye Hoechst 33342 on the sperm by the argon ion Ultra-Violet Laser (~ 350nm) excite, produce blue excitation light (~ 460nm).Because the contained DNA of x sperm is more than y sperm, in conjunction with upper many Hoechst33342, the fluorescence that inspires is just than y sperm strong (Fig. 1).Which flow cytometer tells according to the difference of fluorescence intensity is the x sperm, and which is y sperm.Simultaneously, because nozzle produces the high frequency vibrations, the liquid stream that high-velocity jet goes out has formed the fine droplet that comprises single x sperm or y sperm.After drop is filled with positive charge or negative charge, under the guiding of electrical forces, fall into respectively different collection containers, the x sperm is separated with y sperm, but since traditional flow cytometer for to as if all cells, can't distinguish fast and effectively according to the characteristic of sperm, cause separation efficiency and separation accuracy low.
Summary of the invention
The technical problem that (one) will solve
The technical problem to be solved in the present invention is: how to provide a kind of sperm sorter, to improve efficient and the precision of separated sperm.
(2) technical scheme
For solving the problems of the technologies described above, the invention provides a kind of sperm sorter, described sorter comprises: nozzle, described nozzle lower end is connected with nozzle needle, and the syringe needle of described nozzle needle is wedge shape.
Preferably, described nozzle interior is flat elliptic.
The invention also discloses a kind of sperm sorter, described sorter comprises: laser excitation device, described laser excitation device are that transmitted power is the excitor of the Ultra-Violet Laser of 150mw.
Preferably, the Ultra-Violet Laser of described laser excitation device emission is 355nm.
Preferably, after described sorter adopts 3 eyeglasses through 3 secondary reflections, excite the sperm of dyeing.
The invention also discloses a kind of sperm sorter, described sorter comprises: sensor, the ingress of described sensor is provided with long-length filter.
Preferably, described sensor is provided with two, is located at respectively with the incident direction opposite position of the laser of described laser excitation device emission with the incident direction of the laser of described laser excitation device emission to be 90 degree places.
Preferably, be provided with non-linear amplifier in the described sensor.
Preferably, described sensor is photomultiplier.
Preferably, described photomultiplier is the R928 photomultiplier of Japanese Hamamatsu company.
(3) beneficial effect
The present invention improves on the basis of traditional flow cytometer, so that the efficient of separated sperm and precision have had raising by a relatively large margin, and nearly 3 ~ 5 times of the Speed improving of separation.
Description of drawings
Fig. 1 is the x sperm that excites of traditional loss cell instrument and the fluorescence disparity map of y sperm;
Fig. 2 is 45 degree vertical views according to nozzle lower end in the sperm sorter of one embodiment of the present invention;
Fig. 3 is the front view of nozzle lower end in the sperm sorter shown in Figure 2;
Fig. 4 is the upward view of nozzle lower end in the sperm sorter shown in Figure 2;
Fig. 5 is the side-view of nozzle lower end in the sperm sorter shown in Figure 2;
Fig. 6 is 45 degree upward views of nozzle lower end in the sperm sorter shown in Figure 2;
Fig. 7 is the vertical view of nozzle lower end in the sperm sorter shown in Figure 2;
Fig. 8 is the x sperm that excites of sperm sorter shown in Figure 2 and the fluorescence disparity map of y sperm;
Fig. 9 be in the sperm sorter shown in Figure 2 sensor synoptic diagram is set.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The sperm sorter of present embodiment comprises: nozzle, and the inside of described nozzle is provided with the flat elliptic shower nozzle; Described nozzle lower end is connected with nozzle needle, and the syringe needle of described nozzle needle is wedge shape; The sprinkler selection round pin of traditional flow cytometer, and by testing the tadpole-shaped that is shaped as of sperm, seminal fluid sprays from shower nozzle in sepn process with sheath fluid, sperm can enter into different angles the laser excitation zone, the position that practical laser excites sperm sperm with plane become with laser 45 degree excite into signal the strongest.The difference of X and Y chromosome is rendered as maximum like this, reaches the separation purpose of precise and high efficiency.The sorter of present embodiment is the mode of wedge shape by the pin of change shower nozzle, according to hydromechanical principle, form the power of pincer attack at the sheath fluid on syringe needle both sides, impel sperm to enter shower nozzle with certain orientation, in addition, sorter in the present embodiment has been done corresponding improvement to the lower end of nozzle simultaneously, such as Fig. 2 ~ 7, its inside is conformed to the profile of sperm for the flattened oval type.Carry out the location at shower nozzle like this, join with shower nozzle, shower nozzle is to locate with laser 45 degree.So that laser excitation obtains maximum difference signal, as shown in Figure 8, greatly improved recognition rate by such improvement, thus so that velocity of separation raising.
Described sorter comprises: laser excitation device, described laser excitation device are that transmitted power is the excitor of the Ultra-Violet Laser of 150mw; The Ultra-Violet Laser of described laser excitation device emission is 355nm; Through sperm X and Y chromosome after the laser excitation dyeing 3% difference is arranged, then adopt non-linear amplification processing signals to analyze to it; After described sorter adopts 3 eyeglasses through 3 secondary reflections, excite the sperm of dyeing, described eyeglass adopts 45 degree plane mirrors, and adopts trigonometric expression 3 secondary reflections; In the present embodiment, select the seminal fluid after ultraviolet 355nm laser excitation is dyeed.Here the laser of selecting is the Ultra-Violet Laser of the power 150mw wavelength 355nm of U.S. JDSU company product.Select the reason of 150nm, can be killed at the above power of 150mw through the lot of experiments sperm, and excessively low power influences stimulation effect.And the seminal fluid of dyeing excites the maximum of lower difference signal acquisition at the Ultra-Violet Laser of wavelength 355nm.In order to obtain highly purified laser signal, in the flow cytometer light path, select plane 45 degree high-performance plane speculums, its effect is that the reflection of 355 wavelength is filtered other clutter.Here select 3 eyeglasses to enter the flow cytometer cavity through 3 secondary reflections, excite the sperm of dyeing.
Described sorter comprises: sensor 1, and the ingress of described sensor 1 is provided with long-length filter 2, and with reference to Fig. 9, the fluorescent signal that receives laser excitation in this patent is selected the product R928 photomultiplier of 2 Japanese Hamamatsu companies.Through lot of experiments its putting position for to be the signal acquisition of accepting after the laser excitation at 90 degree places the strongest with the incident direction opposite position of the laser of described laser excitation device emission with the incident direction of the laser of described laser excitation device emission.Fluorescent signal after exciting is selected long-length filter, make LP400 so that the ripple of the following wavelength of fluorescence of 400nm can not shine photomultiplier, reason is that the be stimulated fluorescence of rear generation of karyomit(e) is only useful signal more than 400, and this difference just can manifest obviously.
After photomultiplier receives the fluorescence excitation signal, the process analog to digital conversion is electrical signal, this patent is by being that the photomultiplier ECU adds non-linear amplifying circuit so that signal difference is reflected to signal handling equipment with difference through non-linear amplification at traditional flow cytometer PMT, thereby reaches the purpose of separation.
The present invention improves on the basis of traditional flow cytometer, so that the efficient of separated sperm and precision have had raising by a relatively large margin, and nearly 3 ~ 5 times of the Speed improving of separation.
Above embodiment only is used for explanation the present invention; and be not limitation of the present invention; the those of ordinary skill in relevant technologies field; in the situation that does not break away from the spirit and scope of the present invention; can also make a variety of changes and modification; therefore all technical schemes that are equal to also belong to category of the present invention, and scope of patent protection of the present invention should be defined by the claims.
Claims (10)
1. a sperm sorter is characterized in that, described sorter comprises: nozzle;
The inside of described nozzle is provided with the flat elliptic shower nozzle;
Described nozzle lower end is connected with nozzle needle, and the syringe needle of described nozzle needle is wedge shape.
2. sorter as claimed in claim 1 is characterized in that, described shower nozzle adopts stupalith.
3. a sperm sorter is characterized in that, described sorter comprises: laser excitation device, described laser excitation device are that transmitted power is the excitor of the Ultra-Violet Laser of 150mw.
4. sorter as claimed in claim 3 is characterized in that, the Ultra-Violet Laser of described laser excitation device emission is 355nm.
5. such as claim 3 or 4 described sorters, it is characterized in that, after described sorter adopts 3 eyeglasses through 3 secondary reflections, excite the sperm of dyeing;
Described eyeglass adopts 45 degree plane mirrors, and adopts trigonometric expression 3 secondary reflections.
6. a sperm sorter is characterized in that, described sorter comprises: sensor, the ingress of described sensor is provided with long-length filter.
7. sorter as claimed in claim 6 is characterized in that, described sensor is provided with two, is located at respectively with the incident direction opposite position of the laser of described laser excitation device emission with the incident direction of the laser of described laser excitation device emission to be 90 degree places.
8. such as claim 6 or 7 described sorters, be provided with non-linear amplifier in the described sensor.
9. such as claim 6 or 7 described sorters, it is characterized in that described sensor is photomultiplier.
10. sorter as claimed in claim 9 is characterized in that, described photomultiplier is the R928 photomultiplier of Japanese Hamamatsu company.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103520511A CN103013811A (en) | 2011-09-20 | 2012-09-20 | Sperm sorter |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110280446 | 2011-09-20 | ||
| CN201110280446.0 | 2011-09-20 | ||
| CN2012103520511A CN103013811A (en) | 2011-09-20 | 2012-09-20 | Sperm sorter |
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| CN103013811A true CN103013811A (en) | 2013-04-03 |
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| CN2012103520511A Pending CN103013811A (en) | 2011-09-20 | 2012-09-20 | Sperm sorter |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103540520A (en) * | 2013-09-29 | 2014-01-29 | 大连金弘基种畜有限公司 | Elliptic cone threaded nozzle |
| WO2014047358A1 (en) * | 2012-09-19 | 2014-03-27 | Inguran, Llc | Flow cytometer nozzle tip |
| US9027850B2 (en) | 2012-09-19 | 2015-05-12 | Inguran, Llc | Nozzle assembly for a flow cytometer system and methods of manufacture |
| CN106796171A (en) * | 2014-08-11 | 2017-05-31 | 基因扩散公司 | The apparatus and method of the eukaryotic in transport channel are selected by changing eukaryotic with electromagnetic radiation |
| CN112136034A (en) * | 2018-04-27 | 2020-12-25 | 贝克顿·迪金森公司 | Collection system for flow cytometry sorting samples and methods of use thereof |
| US11668640B2 (en) | 2015-03-06 | 2023-06-06 | Inguran, Llc | Nozzle assembly for a flow cytometry system and methods of manufacture |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04204254A (en) * | 1990-11-30 | 1992-07-24 | Nippon Koden Corp | Cell sorter |
| CN1402831A (en) * | 1999-12-03 | 2003-03-12 | Xy公司 | Improved flow cytometer nozzle and flow cytometer sample handling methods |
| CN1433267A (en) * | 2000-05-09 | 2003-07-30 | Xy公司 | High purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa |
| WO2004104178A2 (en) * | 2003-05-15 | 2004-12-02 | Xy, Inc. | Efficient haploid cell sorting for flow cytometer systems |
| CN101504406A (en) * | 1997-12-31 | 2009-08-12 | Xy公司 | Sex-specific insemination of mammals with low number of sperm cells |
| CN101614731A (en) * | 2003-03-28 | 2009-12-30 | 英格朗公司 | Be used to sort equipment, method and the program of particle and the animal sperm that the sex letter sorting is provided |
-
2012
- 2012-09-20 CN CN2012103520511A patent/CN103013811A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04204254A (en) * | 1990-11-30 | 1992-07-24 | Nippon Koden Corp | Cell sorter |
| CN101504406A (en) * | 1997-12-31 | 2009-08-12 | Xy公司 | Sex-specific insemination of mammals with low number of sperm cells |
| CN1402831A (en) * | 1999-12-03 | 2003-03-12 | Xy公司 | Improved flow cytometer nozzle and flow cytometer sample handling methods |
| CN1433267A (en) * | 2000-05-09 | 2003-07-30 | Xy公司 | High purity X-chromosome bearing and Y-chromosome bearing populations of spermatozoa |
| CN101614731A (en) * | 2003-03-28 | 2009-12-30 | 英格朗公司 | Be used to sort equipment, method and the program of particle and the animal sperm that the sex letter sorting is provided |
| WO2004104178A2 (en) * | 2003-05-15 | 2004-12-02 | Xy, Inc. | Efficient haploid cell sorting for flow cytometer systems |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014047358A1 (en) * | 2012-09-19 | 2014-03-27 | Inguran, Llc | Flow cytometer nozzle tip |
| US9027850B2 (en) | 2012-09-19 | 2015-05-12 | Inguran, Llc | Nozzle assembly for a flow cytometer system and methods of manufacture |
| US9222872B2 (en) | 2012-09-19 | 2015-12-29 | Inguran, Llc | Flow cytometer nozzle tip |
| US9404846B2 (en) | 2012-09-19 | 2016-08-02 | Inguran, Llc | Nozzle assembly for a flow cytometer system and methods of manufacture |
| US9927345B2 (en) | 2012-09-19 | 2018-03-27 | Inguran, Llc | Nozzle assembly for a flow cytometer |
| US10634601B2 (en) | 2012-09-19 | 2020-04-28 | Inguran, Llc | Nozzle assembly for a flow cytometry system and methods of manufacture |
| CN103540520A (en) * | 2013-09-29 | 2014-01-29 | 大连金弘基种畜有限公司 | Elliptic cone threaded nozzle |
| CN106796171A (en) * | 2014-08-11 | 2017-05-31 | 基因扩散公司 | The apparatus and method of the eukaryotic in transport channel are selected by changing eukaryotic with electromagnetic radiation |
| US11668640B2 (en) | 2015-03-06 | 2023-06-06 | Inguran, Llc | Nozzle assembly for a flow cytometry system and methods of manufacture |
| CN112136034A (en) * | 2018-04-27 | 2020-12-25 | 贝克顿·迪金森公司 | Collection system for flow cytometry sorting samples and methods of use thereof |
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Application publication date: 20130403 |