The present application is a U.S. National Stage Application under 35 U.S.C. § 371, based on International Application No. PCT/JP2014/074610, filed Sep. 10, 2014, which claims priority to Japanese Patent Application JP 2013-215736, filed Oct. 16, 2013, the entire contents of each of which is incorporated herein by reference.
The present technique relates to a particle sorting apparatus, a particle sorting method, and a program, more specifically, to a technique of sorting particles based on an analysis result using an optical method or the like.
From the past, an optical measurement method that uses flow cytometry (flow cytometer) has been used for analyzing biologically-relevant micro-particles such as cells, microorganisms, and liposomes. A flow cytometer is an apparatus that irradiates light onto micro-particles flowing through a flow channel formed in a flow cell, a microchip, and the like and detects and analyzes fluorescence and scattered light emitted from the individual micro-particles.
Some flow cytometers include a function of sorting and recovering only micro-particles having a specific property based on an analysis result, and a micro-particle apparatus that sorts cells in particular is called “cell sorter”. In such a cell sorter, in general, a flow cell or a microchip is vibrated by a vibration device or the like to form droplets of a fluid discharged from the flow channel (see Patent Documents 1 and 2).
After the droplets separated from the fluid is imparted with charges of plus (+) and minus (−), a traveling direction thereof is changed by a deflection plate or the like so that the droplets are recovered in a predetermined vessel or the like. Further, from the past, there has also been proposed a technique of distributing particular cells one by one to each reaction site of a base material used in a PCR (Polymerase Chain Reaction) method or the like using a sorting function of a cell sorter (see Patent Document 3).
Patent Document 1: Japanese Patent Translation Publication No. 2007-532874
Patent Document 2: Japanese Patent Application Laid-open No. 2010-190680
Patent Document 3: Japanese Patent Translation Publication No. 2010-510782
However, the particle sorting apparatus of the related art described above has a problem that when particles of different sizes coexist in a sample fluid, the traveling direction of droplets becomes unstable so as not to be distributed to a predetermined vessel or reaction site, thus resulting in lowering of sorting accuracy and sorting efficiency.
In this regard, the present disclosure aims at providing a particle sorting apparatus, a particle sorting method, and a program that are capable of efficiently sorting particles even when sizes of particles to be sorted are not uniform.
According to the present disclosure, there is provided a particle sorting apparatus including: a charge section that imparts charges to at least a part of droplets discharged from an orifice from which a fluid stream is generated; and a charge control section that adjusts a charge amount to be imparted by the charge section according to sizes of particles included in the droplets.
When the particle sorting apparatus further includes a forward-scattered light detection section that irradiates light onto particles flowing through a flow channel and detects forward-scattered light emitted from the particles by the light irradiation, the charge control section may determine, based on a result of the detection by the forward-scattered light detection section, the charge amount to be imparted to the droplets including the particles.
The charge control section may control the charge section to: impart a first charge amount to the droplets including the particles when an intensity of forward-scattered light detected by the forward-scattered light detection section is smaller than a preset threshold value; and impart a second charge amount different from the first charge amount to the droplets including the particles when the intensity of forward-scattered light detected by the forward-scattered light detection section is equal to or larger than the preset threshold value.
In this case, the second charge amount may be set to be larger than the first charge amount.
The charge control section may change the charge amount to be imparted to the droplets including the particles in proportion to an intensity of forward-scattered light detected by the forward-scattered light detection section.
The charge section may include a charge electrode that is provided while being in contact with a sheath fluid and/or a sample fluid flowing through a flow channel, and the charge control section may change a voltage to be applied to the charge electrode to adjust the charge amount to be imparted to the droplets.
The orifice may be formed in a replaceable microchip. In this case, the charge electrode may be provided in a sheath fluid flow channel provided in the microchip.
The particle sorting apparatus according to the present disclosure may further include: an image pickup device that picks up an image of a state of the droplets; and a droplet judgment section that judges the state of the droplets from the image picked up by the image pickup device, and the charge control section may adjust the charge amount when the droplet judgment section judges that an adjustment is necessary.
The droplet judgment section may judge that the adjustment is necessary when a small droplet not including the particles exists around the droplets including the particles.
According to the present disclosure, there is provided a particle sorting method including the steps of: imparting charges to at least a part of droplets discharged from an orifice from which a fluid stream is generated; and adjusting a charge amount to be imparted to the droplets according to sizes of particles included in the droplets.
According to the present disclosure, there is provided a program that causes a charge control section of a particle sorting apparatus to execute a function of adjusting, according to sizes of particles included in droplets discharged from an orifice from which a fluid stream is generated, a charge amount to be imparted to the droplets.
According to the present disclosure, target particles can be efficiently sorted irrespective of the particle sizes. It should be noted that the effects described herein are not necessarily limited, and any effect described in the present disclosure may be obtained.
Hereinafter, embodiments of the present disclosure will be described in detail with reference to the attached drawings. It should be noted that the present disclosure is not limited to the following embodiments. Moreover, the descriptions will be given in the following order.
1. First Embodiment
(Example of Sorting Apparatus that Adjusts Charge Amount Based on Particle Size)
2. Second Embodiment
(Example of Sorting Apparatus that Judges Whether to Perform Adjustment Based on Picked-Up Droplet Image)
First, a particle sorting apparatus according to a first embodiment of the present disclosure will be described.
(Entire Structure of Apparatus)
The particle sorting apparatus 1 of this embodiment sorts and recovers particles based on a result of an analysis using an optical method or the like. As shown in
(Particles)
Particles analyzed and sorted by the particle sorting apparatus 1 of this embodiment widely include biologically-relevant micro-particles such as cells, microorganisms, and liposomes and synthetic particles such as latex particles, gel particles, and industrial particles.
Biologically-relevant micro-particles include chromosomes, liposomes, mitochondria, and organelle (cell organelle) constituting various cells. In addition, cells include plant cells, animal cells, and hemocyte cells. Further, microorganisms include bacteria such as coliform bacillus, viruses such as tobacco mosaic virus, and fungi such as yeast. The biologically-relevant micro-particles may also include biologically-relevant polymer molecules such as nucleic acid, protein, and a complex of those.
On the other hand, industrial particles include particles formed of, for example, an organic polymer material, an inorganic material, and a metal material. As the organic polymer material, polystyrene, styrene divinylbenzene, polymethyl methacrylate, or the like can be used. Moreover, as the inorganic material, glass, silica, a magnetic material, or the like can be used. As the metal material, for example, gold colloid or aluminum can be used. It should be noted that the shape of the particles is generally a sphere, but the shape may be an aspherical shape, and the size, mass, and the like are also not limited in particular.
(Microchip 2)
Formed in the microchip 2 are a sample inlet 22 into which a fluid including particles to be sorted (sample fluid) is introduced, a sheath inlet 23 into which a sheath fluid is introduced, a suction outlet 24 for eliminating clogging and air bubbles, and the like. In the microchip 2, a sample fluid is introduced into the sample inlet 22 to join a sheath fluid introduced into the sheath inlet 23 and is thereafter sent into a sample flow channel to be discharged from an orifice 21 provided at a terminal of the sample flow channel.
Further, a suction flow channel that is in communication with the suction outlet 24 is connected to the sample flow channel. The suction flow channel is used for eliminating, when clogging or air bubble is caused in the sample flow channel, the clogging or air bubble by setting the inside of the sample flow channel to a negative pressure to temporarily cause back-flow. A negative pressure source such as a vacuum pump is connected to the suction outlet 24.
The microchip 2 can be formed of glass and various plastics (PP, PC, COP, PDMS, etc.). The material of the microchip 2 is desirably a material that has permeability with respect to measurement light irradiated from a photodetector to be described later, less auto-fluorescence, and less optical errors due to a small wavelength dispersion.
The microchip 2 can be formed by wet etching or dry etching on a glass substrate or nanoimprint, injection molding, and mechanical processing on a plastic substrate. The microchip 2 can be formed by, for example, sealing a substrate on which a sample flow channel or the like is formed by a substrate formed of the same material or a different material.
(Vibration Device 3)
The vibration device 3 is provided while being in contact with a part of the microchip 2 or as an internal structure of the microchip 2. The vibration device 3 causes the microchip 2 to vibrate at a predetermined frequency and causes minute vibrations with respect to the sheath fluid to form droplets of the fluids (sample fluid and sheath fluid) discharged from the orifice 21, with the result that a fluid stream (flow of droplets) S is generated. As the vibration device 3, a piezo device or the like can be used.
(Charge Section 4)
The charge section 4 imparts positive or negative charges to the droplets discharged from the orifice 21 and is constituted of a charge electrode 41, a voltage source (voltage supply section 42) that applies a predetermined voltage to the charge electrode 41, and the like. The charge electrode 41 is provided while being in contact with the sheath fluid and/or sample fluid flowing through the flow channel and imparts charges to the sheath fluid and/or sample fluid. The charge electrode 41 is inserted into, for example, a charge electrode inlet of the microchip 2.
It should be noted that although the charge electrode 41 is in contact with the sample fluid in
As described above, by imparting positive or negative charges to desired droplets to charge them, the droplets including arbitrary particles can be separated by an electrical force. Moreover, by synchronizing the charge timing of the charge section 4 and the supply voltage with respect to the vibration device 3, it becomes possible to charge only the arbitrary droplets.
(Deflection Plates 5a and 5b)
The deflection plates 5a and 5b change the traveling direction of the droplets in the fluid stream S by an electrical force that operates among charges imparted to the droplets and induce them to predetermined recovery vessels 6a to 6c, and are provided opposed to each other while sandwiching the fluid stream S. Normally-used electrodes can be used as the deflection plates 5a and 5b, for example.
Different positive or negative voltages are applied to the deflection plates 5a and 5b. As a result, as the charged droplets pass the formed electrical field, an electrical force (coulomb force) is generated, and the droplets are drawn to a direction of either one of the deflection plates 5a and 5b. In the particle sorting apparatus 1, it is possible to control the flow of droplets (side stream) drawn by an electrical field by varying the positive/negative or the charge amount of charges to the droplets, with the result that a plurality of mutually-different particles can be sorted at the same time.
(Recovery Vessels 6a to 6c)
The recovery vessels 6a to 6c recover droplets that have passed between the deflection plates 5a and 5b, and a general-purpose plastic tube, glass tube, and the like can be used for experiments. It is favorable for the recovery vessels 6a to 6c to be provided while being replaceable in the apparatus. In addition, the vessel that recovers non-purpose particles out of the recovery vessels 6a to 6c may be coupled to a drainage channel for recovered droplets.
It should be noted that the number and types of recovery vessels to be provided in the particle sorting apparatus 1 are not limited in particular. For example, when more than 3 recovery vessels are arranged, the droplets only need to be induced to any one of the recovery vessels depending on the presence/absence of the electrical force between the deflection plates 5a and 5b and the magnitude of the force so as to be recovered. Moreover, it is also possible to use a base material in which a plurality of reaction sites (wells) are formed in place of the recovery vessels 6a to 6c and distribute particular particles one by one to the reaction sites.
(Charge Control Section 7)
The charge control section 7 adjusts a charge amount to be imparted by the charge section 4 according to sizes of particles included in the droplets. A method of judging a particle size is not limited in particular. For example, the particle size can be judged based on a result of detecting forward-scattered light measured by the photodetector to be described later. In this case, the charge control section 7 can vary the charge amount to be imparted to the droplets based on whether the intensity of forward-scattered light detected by the photodetector is equal to or larger than a specific value (threshold value) or vary the charge amount to be imparted to the droplets in proportion to the intensity of forward-scattered light.
Furthermore, the charge amount to be imparted to the droplets can be adjusted by controlling the voltage supply section 42 to change the voltage to be applied to the charge electrode 41, for example. In this case, it is favorable for a higher voltage to be applied as the size of particles increases. Accordingly, since charges suited to the particle sizes can be imparted, the droplets can be stably induced by the deflection plates 5a and 5b.
(Photodetector)
Also provided in the particle sorting apparatus 1 of this embodiment is a photodetector (not shown) that irradiates light (measurement light) onto a predetermined site of the sample flow channel and detects light (measurement target light) generated from particles flowing through the sample flow channel, for example. The photodetector can be structured similar to a flow cytometry of the related art. Specifically, the photodetector is constituted of a laser light source, an irradiation system constituted of a collective lens and dichroic mirror that collect and irradiate laser light onto particles, a bandpass filter, and the like, and a detection system that detects measurement target light generated from particles due to laser light irradiation.
The detection system is constituted of, for example, a PMT (Photo Multiplier Tube) and an area image pickup device such as a CCD and a CMOS device. It should be noted that the irradiation system and detection system may also be structured either by the same optical path or different optical paths. Moreover, the measurement target light detected by the detection system of the photodetector is light generated from particles due to irradiation of measurement light and may be, for example, forward-scattered light or side-scattered light, various types of scattered light caused by Rayleigh scattering and Mie scattering, fluorescence, and the like.
Of those measurement target light, forward-scattered light becomes an index for evaluating the particle size since its intensity varies in proportion to a surface area of cells. Therefore, it is favorable for the particle sorting apparatus 1 of this embodiment to include a forward-scattered light detection section that detects forward-scattered light so that the charge amount adjustment by the charge control section 7 can be performed with ease.
(Operations)
Next, operations of the particle sorting apparatus 1 of this embodiment, that is, a method of sorting particles using the particle sorting apparatus 1 will be described while taking a case where a charge amount is adjusted based on a detection result of forward-scattered light as an example.
When sorting particles by the particle sorting apparatus 1 of this embodiment, the sample fluid including particles to be sorted is introduced into the sample inlet 22, and the sheath fluid is introduced into the sheath inlet 23. Then, for example, the photodetector detects a particle flow speed (flow rate) and particle intervals simultaneous with a detection of optical properties of particles. The detected optical properties of particles, flow rate, intervals, and the like are converted into electrical signals and output to an overall control section (not shown) of the apparatus.
The laminar flow of the sample fluid and sheath fluid that has passed a light irradiation section of the sample flow channel is discharged to a space outside the microchip 2 from the orifice 21. At this time, the orifice 21 is vibrated by the vibration device 3 so that the fluid to be discharged is formed into droplets. Then, the traveling direction of charged droplets in the sample flow channel is changed by the deflection plates 5a and 5b to be induced to and recovered in the predetermined recovery vessels 6a to 6c based on the detection result obtained by the photodetector.
At this time, in the particle sorting apparatus 1 of this embodiment, the charge amount to be imparted by the charge section 4 is adjusted according to sizes of particles included in the droplets.
Here, the particle group a shown in
Specifically, the charge control section 7 controls the voltage supply section 42 so that the second charge amount becomes larger than the first charge amount, that is, a higher voltage is applied to the droplets including the particles of the particle group b than the droplets including the particles of the particle group a. The charge amount adjustment based on forward-scattered light is not limited to the two-step adjustment, and by setting a plurality of threshold values, the adjustment can be performed while categorizing particles in 3 or more steps. The threshold values can be set arbitrarily by a user, so the adjustment can be performed as appropriate according to cells to be sorted.
Further, the charge amount to be imparted to the droplets including the particles can be changed in proportion to the intensity of forward-scattered light detected by the photodetector without setting a threshold value. In this case, the charge control section 7 controls the voltage supply section 42 so that a higher voltage is applied to droplets including particles having a higher intensity of forward-scattered light.
It is possible to cause the charge control section 7 to perform the charge amount adjustment described above by creating a program for realizing a function of adjusting a charge amount to be imparted to droplets according to sizes of particles included in the droplets and mounting the program in the charge control section 7 of the particle sorting apparatus 1.
It should be noted that although the first embodiment above exemplifies the case of using the microchip 2, the present disclosure is not limited thereto, and the same effect can be obtained even when a flow cell is used in place of the microchip 2.
As described above in detail, since the charge amount to be imparted by the charge section is adjusted according to sizes of particles included in the droplets in the particle sorting apparatus of this embodiment, the traveling direction of the droplets can be stabilized irrespective of the particle sizes. As a result, the particles can be efficiently sorted even when the sizes of particles to be sorted, that are included in the sample fluid, are not uniform.
Next, a particle sorting apparatus according to a second embodiment of the present disclosure will be described.
(Image Pickup Device (Camera) 8)
The image pickup device (camera) 8 picks up an image of a state of droplets that have passed the deflection plates 5a and 5b. It should be noted that for the image pickup of droplets, various image pickup devices such as a photoelectric conversion device can be used in addition to image pickup apparatuses such as a CCD and a CMOS camera. Moreover, a movement mechanism for changing a position can be provided in the camera 8. In addition, a light source (not shown) that illuminates a photograph area may be provided with the camera 8 in the particle sorting apparatus 11 of this embodiment.
(Droplet Judgment Section 9)
The droplet judgment section 9 judges a state of droplets based on an image picked up by the camera 8 and outputs the result to the charge control section 7. The droplet judgment section 9 judges that an adjustment is necessary when there is a small droplet not including a particle (side strip) around the droplet including a particle, for example.
(Droplet Judgment Step)
First, charged droplets are photographed by the camera 8.
In this regard, in the particle sorting apparatus 11 of this embodiment, states of the droplets 82a to 82c are photographed by the camera 8, and whether the side strips 83 are generated is checked by the droplet judgment section 9. The method of checking the side strips 83 by the droplet judgment section 9 is not limited in particular. For example, whether the side strips 83 are generated can be checked by setting a judgment area 81 including the droplet 82a and carrying out image recognition processing on the judgment area 81.
As a result, when confirmed that the side strip 83 exists, the droplet judgment section 9 judges that the charge amount needs to be adjusted and outputs the judgment result to the charge control section 7. Accordingly, the charge control section 7 adjusts the charge amount. For example, when the side strip 83 is more on the outside than the expected position, the charge amount is reduced by judging that the charge is too strong and in an opposite case, increases the charge amount by judging that the charge is too weak.
Since the particle sorting apparatus 11 of this embodiment judges the droplet state based on the image 80 taken by the camera 8 to judge whether the charge amount is to be adjusted, an adjustment that matches an actual condition can be performed.
It should be noted that the structures and effects of the particle sorting apparatus of this embodiment other than those described above are the same as those of the first embodiment described above.
Furthermore, the present disclosure may also take the following structures.
a charge section that imparts charges to at least a part of droplets discharged from an orifice from which a fluid stream is generated; and
a charge control section that adjusts a charge amount to be imparted by the charge section according to sizes of particles included in the droplets.
a forward-scattered light detection section that irradiates light onto particles flowing through a flow channel and detects forward-scattered light emitted from the particles by the light irradiation,
in which the charge control section determines, based on a result of the detection by the forward-scattered light detection section, the charge amount to be imparted to the droplets including the particles.
in which the charge control section controls the charge section to:
impart a first charge amount to the droplets including the particles when an intensity of forward-scattered light detected by the forward-scattered light detection section is smaller than a preset threshold value; and
impart a second charge amount different from the first charge amount to the droplets including the particles when the intensity of forward-scattered light detected by the forward-scattered light detection section is equal to or larger than the preset threshold value.
in which the second charge amount is larger than the first charge amount.
in which the charge control section changes the charge amount to be imparted to the droplets including the particles in proportion to an intensity of forward-scattered light detected by the forward-scattered light detection section.
the charge section includes a charge electrode that is provided while being in contact with a sheath fluid and/or a sample fluid flowing through a flow channel; and
the charge control section changes a voltage to be applied to the charge electrode to adjust the charge amount to be imparted to the droplets.
in which the orifice is formed in a replaceable microchip, and the charge electrode is provided in a sheath fluid flow channel provided in the microchip.
an image pickup device that picks up an image of a state of the droplets; and
a droplet judgment section that judges the state of the droplets from the image picked up by the image pickup device,
in which the charge control section adjusts the charge amount when the droplet judgment section judges that an adjustment is necessary.
in which the droplet judgment section judges that the adjustment is necessary when a small droplet not including the particles exists around the droplets including the particles.
imparting charges to at least a part of droplets discharged from an orifice from which a fluid stream is generated; and
adjusting a charge amount to be imparted to the droplets according to sizes of particles included in the droplets.
a function of adjusting, according to sizes of particles included in droplets discharged from an orifice from which a fluid stream is generated, a charge amount to be imparted to the droplets.
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2013-215736 | Oct 2013 | JP | national |
Filing Document | Filing Date | Country | Kind |
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PCT/JP2014/074610 | 9/10/2014 | WO | 00 |
Publishing Document | Publishing Date | Country | Kind |
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WO2015/056516 | 4/23/2015 | WO | A |
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2011-509075 | Mar 2011 | JP |
2011-232033 | Nov 2011 | JP |
2011-237201 | Nov 2011 | JP |
4805417 | Nov 2011 | JP |
2012-047464 | Mar 2012 | JP |
2013-210264 | Oct 2013 | JP |
2013-210270 | Oct 2013 | JP |
2015-152439 | Aug 2015 | JP |
WO 2001002836 | Jan 2001 | WO |
WO 2010095391 | Aug 2010 | WO |
WO 2010129787 | Nov 2010 | WO |
WO 2010140460 | Dec 2010 | WO |
WO 2013145905 | Oct 2013 | WO |
WO 2014115409 | Jul 2014 | WO |
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Number | Date | Country | |
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20160266027 A1 | Sep 2016 | US |