JP2013210292A - Microparticle separation device and control method of microparticle separation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a control method of a device capable of simply constituting an optical detection system in a microchip type microparticle separation device.SOLUTION: There is provided a control method of a microparticle separation device including a step of changing distance between a microchip in which a flow channel through which fluid is conducted, and an orifice for generating a fluid stream at one end of the flow channel are formed, and an objective lens which condenses and irradiates laser to an upstream part of the orifice of the flow channel. The control method includes the steps of: generating the fluid stream; detecting the generated fluid stream; and changing a relative position between the microchip and the objective lens to move the objective lens to a focal position to the microchip after these procedures.

Description

  The present technology relates to a fine particle sorting device and a control method for the fine particle sorting device. More specifically, the present invention relates to a control method of a microchip type microparticle sorting apparatus, which can prevent water droplets from being attached to an objective lens constituting an optical system for detecting optical characteristics of microparticles.

  2. Description of the Related Art A microparticle sorting device (for example, a flow cytometer) that detects the characteristics of microparticles such as cells optically, electrically, or magnetically and separates and collects only microparticles having predetermined characteristics is known.

  In cell sorting in a flow cytometer, first, a fluid stream (a laminar flow of a sample liquid containing cells and a sheath liquid) is generated from an orifice formed in a flow cell or a microchip, and vibration is applied to the orifice to liquefy the fluid stream. Drops and gives charge to the droplets. Then, the moving direction of the droplet containing the cells discharged from the orifice is electrically controlled, and the target cells having desired characteristics and the other non-target cells are recovered in separate recovery containers.

  For example, in Patent Document 1, as a microchip type flow cytometer, “a flow path through which a liquid containing microparticles flows, an orifice for discharging the liquid flowing through the flow path to a space outside the chip, , A vibrating element for ejecting liquid droplets at an orifice, a charging means for imparting electric charges to the ejected liquid droplets, and a microparticle flowing through the flow path Optical detection means for detecting optical characteristics, a counter electrode disposed opposite to the moving liquid droplet in the direction of movement of the liquid droplet discharged to the space outside the chip, and passing between the counter electrodes And a microparticle sorting device including two or more containers for collecting the droplets.

JP 2010-190680 A

  In the fine particle sorting apparatus, in order to detect the optical characteristics of the fine particles flowing through the flow channel formed in the flow cell or the microchip with high sensitivity, a device for the configuration of the optical detection system has been devised.

  The main object of the present technology is to provide a method for controlling an apparatus in which an optical detection system can be simply configured, particularly in a microchip type microparticle sorting apparatus.

In order to solve the above problems, the present technology provides a microchip having a flow path through which a fluid flows, an orifice that generates a fluid stream at one end of the flow path, and an upstream portion of the orifice in the flow path And a control method for the fine particle sorting apparatus, including a procedure for changing the distance between the objective lens for condensing and irradiating the laser with the objective lens.
The control method includes a procedure for generating the fluid stream, a procedure for detecting the generated fluid stream, and after these procedures, changing the relative positions of the microchip and the objective lens, Moving to a focal position relative to the microchip. Prior to generation of the fluid stream, the distance between the microchip and the objective lens is maintained greater than the working distance of the objective lens relative to the microchip. By moving the objective lens to the focal position after the fluid stream has been stably ejected, water droplets will not adhere to the objective lens even if water droplets are generated at the orifice when the fluid stream begins to exit. .
Further, in this control method, the relative position between the microchip and the objective lens is changed, the distance between the microchip and the objective lens is changed to be larger than the working distance, and then the orifice is used. Stopping the injection of the fluid stream. Accordingly, even when water droplets are generated at the orifice when the ejection of the fluid stream is stopped, the water droplets can be prevented from adhering to the objective lens.
In this control method, the fluid stream can be detected by detecting a bright spot in an image of the fluid stream by image recognition.

Further, according to the present technology, a microchip in which a flow path through which a fluid flows and an orifice that generates a fluid stream is formed at one end of the flow path is mounted, and a laser is installed in an upstream portion of the orifice in the flow path. Microparticles comprising: an objective lens that collects and irradiates the light; a stream detection unit that detects the fluid stream ejected from the orifice; and a position adjustment unit that changes a distance between the microchip and the objective lens. A preparative device is provided.
In this fine particle sorting apparatus, the distance between the microchip and the objective lens can be changed, and the stream detection unit is provided, so that the objective lens is moved from the orifice until the fluid stream is ejected. It can be kept away from and close to the focal position after the fluid stream is emitted.
In this microparticle measurement device, the stream detection unit includes a camera that images the fluid stream, and a fluid stream detection light source that irradiates the fluid stream with light.
The fine particle measuring apparatus includes a control unit that detects a bright spot in an image captured by the camera by image recognition, and outputs a signal to the position adjusting unit to adjust the distance.
In this fine particle measuring apparatus, it is preferable that a member for preventing movement of the fluid from the orifice toward the objective lens is disposed between the orifice and the objective lens. In this case, it is preferable that the member has absorbability with respect to the fluid.

In the present technology, “microparticles” widely include living body-related microparticles such as cells, microorganisms, and liposomes, or synthetic particles such as latex particles, gel particles, and industrial particles.
Biologically relevant microparticles include chromosomes, liposomes, mitochondria, organelles (organelles) that constitute various cells. Cells include animal cells (such as blood cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biologically relevant microparticles may include biologically relevant polymers such as nucleic acids, proteins, and complexes thereof. The industrial particles may be, for example, an organic or inorganic polymer material, a metal, or the like. Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials include glass, silica, magnetic materials, and the like. Metals include gold colloid, aluminum and the like. The shape of these fine particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

  According to the present technology, there is provided a method for controlling an apparatus that can simply configure an optical detection system in a microchip-type fine particle sorting apparatus.

It is a figure for demonstrating the structure of the microparticle fractionation apparatus 1 (flow cytometer 1) based on 1st embodiment of this technique comprised as a microchip type | mold flow cytometer. It is a figure for demonstrating the structure of the chip loading module 11 of the flow cytometer 1. FIG. 2 is a diagram for explaining an example of a configuration of a microchip 2 that can be mounted on a flow cytometer 1. FIG. 4 is a diagram for explaining a configuration of an orifice 21 of the microchip 2. FIG. It is a figure for demonstrating the objective lens 14 which comprises the light irradiation detection part of the flow cytometer 1. FIG. 4 is a flowchart for explaining position control in the flow cytometer 1 until analysis is started after completion of mounting the microchip 2 on the chip loading module 11. It is a figure explaining the position of the microchip 2 and the objective lens 14 in each position control step of the flow cytometer 1. It is a flowchart for demonstrating position control in the flow cytometer 1 after the completion of analysis. FIG. 6 is a diagram for explaining a configuration of a water droplet blocking plate 15.

Hereinafter, preferred embodiments for carrying out the present technology will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this technique, and, thereby, the scope of this technique is not interpreted narrowly. The description will be made in the following order.

1. (1) Chip loading module (2) Microchip (3) Light irradiation detection unit (4) Position adjustment unit (5) Stream detection unit (6) Deflection plate (7) Collection tube holder ( 8) Control unit 2. Control of microparticle sorting device (1) Position control at start of analysis (1-1) Chip position initialization step S ₁
(1-2) Fluid stream generation step S ₂
(1-3) stream detection step _{S 3}
(1-4) Chip position moving step S ₄
(2) Position control at the end of analysis (2-1) Chip position return step S ₅
(2-2) fluid stream stop step _{S 6}

1. Device Configuration of Fine Particle Sorting Device FIG. 1 shows the configuration of a micro particle sorting device 1 (hereinafter also referred to as “flow cytometer 1”) according to the first embodiment of the present technology configured as a microchip type flow cytometer. FIG. FIG. 2 is a schematic diagram illustrating the configuration of the chip loading module of the flow cytometer 1. 2A shows a state where the microchip is in the insertion position, and FIG. 2B shows a state where the microchip is in the holding position.

  The flow cytometer 1 includes a pair of chip loading modules 11 that hold the microchip 2 and a pair of fluids S (or droplets that are ejected) ejected from the orifice 21 of the microchip 2 so as to face each other. Deflection plates 12 and 12 and a collection tube holder 13 in which a collection tube 3 that receives the fluid stream S is disposed. Furthermore, the flow cytometer 1 includes a stream detection unit 16 that detects the fluid stream S ejected from the orifice 21, a light irradiation detection unit and a position adjustment unit that are not shown in FIGS. 1 and 2. The light irradiation detection unit functions to detect optical characteristics of cells flowing through the flow path of the microchip 2. Further, the position adjustment unit functions to change the distance between the objective lens constituting the light irradiation detection unit and the microchip 2. Hereafter, each structure is demonstrated in order.

(1) Chip Loading Module The chip loading module 11 transports the microchip 2 inserted into the inside through the chip insertion port 112 from the insertion position (see FIG. 2A) to the holding position (see FIG. 2B). A loading unit 111 is included. The conveyance direction of the microchip 2 from the insertion position to the holding position is indicated by the positive Y-axis direction.

  Inside the loading unit 111, a rotation roller (not shown) is arranged as a conveyance mechanism to convey the microchip 2 inserted from the chip insertion port 112 by sandwiching and rotating. In the fine particle sorting apparatus according to the present technology, the transport mechanism of the loading unit 111 is not limited to that using a rotating roller.

  The chip loading module 11 also includes a liquid feeding connector section 114 that supplies a sample liquid containing cells, a sheath liquid, and the like to the held microchip 2. A sample line 115 can be connected to the liquid feeding connector portion 114. In addition, a sheath line from the sheath liquid tank and a suction line from a negative pressure source such as a vacuum pump are connected to the liquid feeding connector portion 114. In FIG. 2, the sheath line and the suction line are illustrated as a sheath / suction line 116 accommodated in one pipe. In the fine particle sorting apparatus according to the present technology, the suction line is not an essential component.

  Further, the chip loading module 11 applies vibration to the orifice 21 formed in the microchip 2 to impart a charge to the ejected liquid droplets and the chip vibration unit that converts the fluid stream S into liquid droplets and ejects them. A charging part (both not shown).

(2) Microchip FIGS. 3 and 4 show an example of the microchip 2 that can be mounted on the flow cytometer 1. FIG. 3A is a schematic top view, and FIG. 3B is a schematic cross-sectional view corresponding to a cross section along line PP in FIG. 4A and 4B are schematic views showing the configuration of the orifice 21 of the microchip 2. FIG. 4A is a top view, FIG. 4B is a sectional view, and FIG. 4C is a front view. FIG. 4B corresponds to a PP cross section in FIG.

  The microchip 2 is formed by bonding the substrate layers 2a and 2b on which the sample channel 22 is formed. Formation of the sample flow path 22 to the substrate layers 2a and 2b can be performed by injection molding of a thermoplastic resin using a mold. As the thermoplastic resin, known plastics can be employed as materials for conventional microchips such as polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethylsiloxane (PDMS).

  The sample liquid is introduced into the sample inlet 23 from the liquid feeding connector part 114, joins with the sheath liquid introduced from the liquid feeding connector part 114 into the sheath inlet 24, and is fed through the sample flow path 22. After the sheath liquid introduced from the sheath inlet 24 is divided and fed in two directions, the sample liquid is sandwiched from the two directions at the junction with the sample liquid introduced from the sample inlet 23. Join. As a result, a three-dimensional laminar flow in which the sample liquid laminar flow is located at the center of the sheath liquid laminar flow is formed at the junction.

  Reference numeral 25 denotes a suction flow path for eliminating clogging or bubbles by applying a negative pressure in the sample flow path 22 to temporarily reverse the flow when clogging or bubbles are generated in the sample flow path 22. . A suction outlet 251 connected to a negative pressure source such as a vacuum pump is formed at one end of the suction flow path 25 via the liquid feed connector portion 114, and the other end is connected to the sample flow path 22 at the communication port 252. Yes.

  In the three-dimensional laminar flow, the narrowing portions 261 (see FIG. 3) and 262 (see FIG. 4) formed so that the area of the vertical cross section with respect to the liquid feeding direction is gradually or gradually reduced from the upstream to the downstream of the liquid feeding direction. The laminar flow width is narrowed down. Thereafter, the three-dimensional laminar flow is discharged as a fluid stream S (see FIG. 1) from an orifice 21 provided at one end of the flow path. In FIG. 1, the discharge direction of the fluid stream S from the orifice 21 is indicated by the positive Y-axis direction.

  A connecting portion of the sample channel 22 to the orifice 21 is a straight portion 27 formed in a straight line. The straight portion 27 functions to eject the fluid stream S straight from the orifice 21 in the positive direction of the Y axis.

  The fluid stream S ejected from the orifice 21 is formed into droplets by the vibration applied to the orifice 21 by the tip vibration unit. The orifice 21 opens in the direction of the end face of the substrate layers 2a and 2b, and a notch 211 is provided between the opening position and the end face of the substrate layer. The notch 211 is formed by notching the substrate layers 2 a and 2 b between the opening position of the orifice 21 and the substrate end surface so that the diameter L of the notch 211 is larger than the opening diameter l of the orifice 21. (See FIG. 4C). The diameter L of the notch 211 is desirably formed to be twice or more larger than the opening diameter l of the orifice 21 so as not to hinder the movement of the droplets discharged from the orifice 21.

(3) Light Irradiation Detection Unit The flow cytometer 1 includes a light irradiation detection unit for detecting optical characteristics of cells flowing through the sample flow path 22 of the microchip 2 held in the microchip loading module 11. Prepare. The detection of the optical characteristics of the cells is performed, for example, between the narrowing part 261 and the narrowing part 262 of the sample channel 22.

  The light irradiation detection unit is an irradiation made of a laser light source and an objective lens, a dichroic mirror, a band-pass filter, or the like that focuses and irradiates the laser between the narrowing part 261 and the narrowing part 262 of the sample flow path 22. Includes systems.

FIG. 5 shows only the objective lens together with the microchip 2 in the configuration of the irradiation system. Objective lens 14, the laser L ₁ from the laser light source (not shown), the condenser-irradiated to cells P flowing through are arranged in a row the sample channel 22 to the center of the three-dimensional laminar flow. The laser irradiation direction with respect to the microchip 2 is indicated by the positive Z-axis direction. In the Figure, for convenience, it shows only cells P which laser L ₁ is irradiated. In the figure, reference numeral 15 denotes a water droplet blocking plate for preventing adhesion of water droplets to the objective lens 14. The water droplet prevention plate 15 will be described in detail later.

The light irradiation detection unit also includes a detection system for detecting a target light generated from the cells P by irradiation of the laser L _1. The detection system includes a PMT (photo multiplier tube), an area imaging device such as a CCD or a CMOS device, and the like. The objective lens 14 described above can also function to collect measurement target light. The measurement target light detected by the detection system is light generated from the cell P by irradiation of the measurement light. For example, forward scattered light, side scattered light, scattered light such as Rayleigh scattering or Mie scattering, fluorescence, etc. can do. These measurement target lights are converted into electric signals and used for determining the optical characteristics of the cell P.

(4) Position Adjustment Unit Furthermore, the flow cytometer 1 also includes a position adjustment unit that changes the distance between the microchip 2 and the objective lens 14 (see reference sign D in FIG. 5). The position adjusting unit changes the position of the chip loading module 11 that holds the microchip 2 and / or the objective lens 14 in the optical axis direction (Z-axis direction) of the laser. The position adjustment unit can be, for example, a Z-axis stepping motor provided in one or both of the chip loading module 11 and the objective lens 14. FIG. 1 shows a configuration in which a Z-axis stepping motor 113 is provided in the chip loading module 11. The position adjustment unit includes a sensor such as a Hall element for detecting the position of the chip loading module 11 or the objective lens 14.

(5) Stream Detection Unit The flow cytometer 1 includes a CCD camera 161 that images the fluid stream S. Further, the flow cytometer 1 includes a fluid stream S and the fluid stream detector light source 162 for irradiating a laser L _2, and Z-axis motor 163, the stream detector unit 16 configured to include a (see FIG. 1).

Fluid stream detector light source 162 is preferably configured to be parallel to emit the laser L ₂ in the opposing direction of the deflection plates 12, 12 (X-axis direction). Further, the stream detection unit 16, the Z-axis motor 163, movable in the direction perpendicular to the injection direction of the fluid stream S (Y-axis direction) and the direction of the laser L ₂ (X-axis direction) (Z-axis direction) It is preferred that An arrow f in FIG. 1 indicates the moving direction of the stream detection unit 16. The CCD camera 161 may be an imaging means such as a line sensor, a photoelectric conversion element such as a single-plate photodiode, and the fluid stream detection light source 42 is not limited to a laser light source such as an LED or LD, for example, It is also possible to use a xenon light or an incandescent light source.

In the flow cytometer 1, the trajectory of the fluid stream S ejected from the orifice 21 differs depending on the individual difference of the mounted microchip 2, and the position of the fluid stream S changes in the Z-axis direction ( And the X-axis direction). In addition, even in the same microchip 2, the position of the fluid stream S may change in the same manner for each measurement. The stream detection unit 16, the laser L ₂ emitted in the X-axis direction, by movable in the Z axis direction, even if the position in the Z axis direction of the fluid stream S is changed, the laser to the position change the L ₂ it is possible to follow.

(6) Deflection Plates Reference numerals 12 and 12 in FIG. 1 denote a pair of deflection plates disposed opposite to each other with the fluid stream S (or droplets to be discharged) ejected from the orifice 21 interposed therebetween. The deflecting plates 12 and 12 are configured to include an electrode that controls the moving direction of the droplet discharged from the orifice 21 by an electric acting force with the electric charge applied to the droplet. In FIG. 1, the opposing direction of the polarizing plates 12 and 12 is indicated by the X-axis direction.

(7) Collection tube holder In the flow cytometer 1, the fluid stream S (or droplets thereof) is a plurality of collection tubes (collection containers) arranged in a row in the opposing direction (X-axis direction) of the deflection plates 12 and 12. ) Accepted by any of 3 The collection tube 3 may be a general-purpose plastic tube or glass tube for research use. Although the number of the collection tubes 3 is not particularly limited, the case where five collection tubes are installed is illustrated here. The fluid stream S generated from the orifice 21 is guided and collected in any one of the five collection tubes 3 depending on the presence / absence of the electric acting force between the deflecting plates 12 and 12 or the magnitude thereof. The collection tube 3 is installed in the collection tube holder 13 in a replaceable manner.

(8) Control unit In addition to the above-described configuration, the flow cytometer 1 includes a data analysis unit for determining optical characteristics of cells, a tank unit for storing a sample solution and a sheath solution, and so far, provided in a normal flow cytometer. The control part etc. for controlling each structure demonstrated in 1 are provided.

  The control unit can be configured by a general-purpose computer including a CPU, a memory, a hard disk, and the like. The hard disk stores an OS and a program for executing each step of position control described below. The flow cytometer 1 may include an input device such as a keyboard and an output device such as a display and a speaker as a user interface.

2. Control of Fine Particle Sorting Device (1) Position Control at Start of Analysis FIG. 6 is a flowchart of position control in the flow cytometer 1 from the completion of mounting the microchip 2 to the chip loading module 11 until analysis is started. Indicated by The control step includes a procedure of “tip position initialization step S ₁ ”, “fluid stream generation step S ₂ ”, “stream detection step S ₃ ”, and “tip position movement step S ₄ ”. Hereinafter, each procedure will be described.

(1-1) Chip position initialization step S ₁
After the completion of mounting of the microchip 2 on the chip loading module 11, when an instruction to start analysis is input by the user, the control unit outputs a signal to the position adjustment unit, and the distance between the microchip 2 and the objective lens 14. D is set to an initial value D ₀ (see FIG. 7A). Alternatively, the control unit, the distance D is sure that the initial value D _0. Distance setting to the initial value D ₀ of D can be carried out by changing either at least one of the position of the tip loading module 11 and / or the objective lens 14 for holding the microchip 2, the chip loading below A case where only the position of the module 11 (synonymous with the position of the microchip 2) is changed will be described as an example. The position of the chip loading module 11 or the objective lens 14 is detected by a sensor provided in the position adjustment unit and output to the control unit.

(1-2) Fluid stream generation step S ₂
In this step S ₂ , the liquid feeding connector unit 114 starts feeding the sample liquid and the sheath liquid to the sample inlet 23 and the sheath inlet 24 of the microchip 2, and the fluid stream S is ejected from the orifice 21. The control unit outputs a signal to the liquid feeding connector unit 114 to start feeding the sample liquid and the sheath liquid. Further, the control unit starts exciting the orifice 21 from the tip exciting unit to make the fluid stream S into droplets.

At this time, when the fluid stream S starts to emerge from the orifice 21, water droplets W may be formed on the orifice 21 (see FIG. 7B). If the water droplets W scatter and adhere to the objective lens 14, the objective lens 14 becomes dirty, and there is a risk that detection of the optical characteristics of the cells may become impossible or detection accuracy may be reduced. The same problem may occur when water droplets W adhere to the components of the light irradiation detection unit arranged around the objective lens 14. Therefore, flow cytometer in meter 1, the initial value D ₀ of the distance D between the microchip 2 and the objective lens 14, the scattered droplets W is the objective lens 14 and a sufficient distance so as not be reached in the vicinity thereof Is set. The initial value D ₀ can be appropriately set according to the type of sample liquid to be analyzed, the liquid feeding pressure of the sample liquid and the sheath liquid, or the opening diameter l of the orifice 21.

(1-3) stream detection step _{S 3}
In this step S _3, the control unit can move the stream detection unit 16 is driven by a Z axis motor 163 in the Z-axis direction (see FIG. 1 arrow f), the fluid stream emitted from the orifice 21 by the CCD camera 161 Imaging of S (or droplets to be discharged) is performed. The movement of the stream detection unit 16, CCD camera 161 and the fluid stream detection light source 162 is also moved in the Z axis direction, the laser L ₂ emitted from the fluid stream detector light source 162 is scanned in the Z-axis direction.

The image of the fluid stream S picked up by the CCD camera 161 is output to the control unit, and the control unit detects a bright spot in the image. Moving the stream detection unit 16 in the Z-axis direction, when the laser L ₂ continue to scan in the same direction, the laser L ₂ irradiates the fluid stream S in some position. FIG. 1 shows the position of the stream detection unit 16 when the laser L ₂ is at the irradiation position of the fluid stream S.

When the laser L ₂ is at the irradiation position of the fluid stream S, the laser L ₂ irradiation position of the fluid stream S is detected as a high-luminance pixel (bright spot) in the image of the fluid stream S imaged by the CCD camera 161. The On the other hand, when the laser L ₂ is not at the irradiation position of the fluid stream S, a portion illuminated by the laser L ₂ does not occur in the fluid stream S. Not detected.

  That is, by detecting the bright spot in the image of the fluid stream S, the fluid stream S (or ejected droplets) ejected from the orifice 21 can be detected, and the stable fluid stream S is formed in the orifice 21. Can be confirmed. When the fluid stream S cannot be detected, the control unit outputs characters, images, sounds, and the like representing an error from the output device and presents them to the user, and stops the feeding of the sample liquid and the sheath liquid.

(1-4) Chip position moving step S ₄
When the emission of the fluid stream S from the orifice 21 is confirmed by detecting the bright spot in the image of the fluid stream S, the control unit outputs a signal to the position adjusting unit, and the microchip 2, the objective lens 14, Is set to a working distance D _f (D _f <D ₀ ) (see FIG. 7C). In this step S _4, since already a stable state in which the fluid stream S is formed, the water drop W orifice 21 can not exist.

The working distance D _f is a distance at which the laser L ₁ focused by the objective lens 14 is focused on the cell P flowing through the sample flow path 22. Figure 5 shows a case where the microchip 2 and the objective lens 14 is positioned at the working distance D _f.

Working distance D _f is previously set in accordance with the lens characteristic of the objective lens 14 may be a stored value. Alternatively, the working distance _Df is a value that is optimized each time so that the detection intensity of the measurement target light from the standard sample (calibration beads) sent to the sample flow path 22 prior to the cell P is maximized. May be. After execution of step S _4, the flow cytometer 1 starts analysis of cell P.

As described above, the flow cytometer 1, since it is detected that stable fluid stream S is injected from the orifice 21, the working distance of the microchip 2 and the objective lens 14 distance between them from an initial value D ₀ It is moved to become _Df . The initial value D ₀ is set to be sufficiently large with respect to the working distance D _f. That is, in the flow cytometer 1, if the fluid stream S is stably emitted from the orifice 21, the objective lens 14 is kept away from the microchip 2, and is brought close to the focal position after the fluid stream S is emitted. . For this reason, in the flow cytometer 1, even when a water droplet W (see FIG. 7B) is generated in the orifice 21 when the fluid stream S starts to be emitted from the orifice 21, the water droplet W adheres to the objective lens 14 and its periphery. Or scattering to the objective lens 14 and its periphery.

To prevent adhesion of water droplets W to the objective lens 14, the initial value D ₀ is preferably set sufficiently large for the working distance D _f. The initial value _{D 0,} for example 1mm or more.

(2) Position Control at the End of Analysis FIG. 8 is a flowchart showing position control steps in the flow cytometer 1 after the analysis is completed. The control step includes a procedure of “tip position return step S ₅ ” and “fluid stream stop step S ₆ ”. Hereinafter, each procedure will be described.

(2-1) chip position return step _{S 5}
At the end of the analysis, the control unit outputs a signal to the position adjusting unit to return the chip loading module 11 to a position where the distance D between the microchip 2 and the microchip 2 becomes the initial value D ₀ from the working distance D _f . (See FIG. 7A). In this step S _5, a stable state of the fluid stream S is injected is maintained from the orifice 21

(2-2) fluid stream stop step _{S 6}
When the sensor detects that the position of the objective lens 14 has returned to the initial position, the control unit stops the supply of the sample liquid and the sheath liquid by the liquid feeding connector unit 114, and the fluid stream S from the orifice 21 is stopped. Ejection (and droplet discharge) is stopped.

Even when the ejection of the fluid stream S from the orifice 21 is stopped, water droplets W may be formed in the orifice 21 (see FIG. 7B). If the water droplets W scatter and adhere to the objective lens 14, the objective lens 14 becomes dirty, and there is a risk that detection of the optical characteristics of the cells may be impossible or detection accuracy may be reduced at the next measurement. Further, it is necessary to clean the objective lens 14 before the next measurement. Therefore, the flow cytometer 1, the distance D between the microchip 2 and the objective lens 14, after splashing water droplets W has changed a sufficient distance D ₀ as not to reach the objective lens 14 and the periphery thereof, The injection of the fluid stream S is stopped. After execution of the step S _6, the flow cytometer 1 includes a standby mode, and outputs text and images prompting the extraction of the microchip 2 when necessary, sound etc. from the output device, is presented to the user.

In order to prevent the water droplet W generated in the orifice 21 from adhering to the objective lens 14, a large aperture lens that can increase the working distance D _f (working distance) is used as the objective lens 14, or the microchip 2 and the objective lens 14 are used. It is also conceivable to perform gel coupling. However, when a large aperture lens is used, the device becomes expensive and large. In addition, gel coupling is not suitable for a microchip type flow cytometer that assumes microchip replacement (disposable use).

  On the other hand, in the flow cytometer 1, the distance D between the microchip 2 and the objective lens 14 can be changed, so that a simple device configuration employing an inexpensive lens with a small working distance can be used. Further, it is possible to prevent the water droplet W from adhering to the objective lens 14. As a result, the flow cytometer 1 can perform maintenance-free and highly accurate analysis.

  In order to prevent the water droplet W generated in the orifice 21 from adhering to the objective lens 14, the flow cytometer 1 includes a water droplet blocking plate 15 (see FIG. 5) in addition to or instead of the above-described position control. It may be. The water droplet blocking plate 15 is disposed between the microchip 2 and the objective lens 14 and prevents the water droplet W generated at the orifice 21 from moving to the objective lens 14 and its periphery. The shape of the water droplet blocking plate 15 is not particularly limited as long as the water droplet W can be prevented from scattering from the orifice 21 to the objective lens 14, and may not be a plate-like member. In order to effectively prevent the water droplets W from scattering to the objective lens 14 and the periphery thereof, the water droplet blocking plate 15 is a member having absorptivity for the solvent of the sample liquid and the sheath liquid (usually water). preferable. As the absorbent member, absorbent paper, non-woven fabric, or the like can be used, and these may be disposed interchangeably between the microchip 2 and the objective lens 14. By using absorbent paper or non-woven fabric that can absorb the water droplets W by capillary action, the water droplets W can be moved to the water droplet blocking plate 15 with priority over the objective lens 14.

  An example of a suitable shape of the water droplet blocking plate 15 is shown in FIG. Reference numeral 153 denotes a non-woven fabric attached to the chip side surface 151 of the water droplet blocking plate 15 facing the microchip 2. The chip side surface 151 is formed with a V-shaped groove 154 from which scattering toward the objective lens 14 is prevented and the water droplets W accumulated on the surface flow downward. The water droplet blocking plate 15 is preferably grounded to prevent charging. This is because if the water droplet blocking plate 15 is charged, the trajectory of the fluid stream S ejected from the orifice 21 may be affected.

The control method of the fine particle sorting apparatus according to the present technology can also be configured as follows.
(1) A microchip in which a flow path through which a fluid flows, an orifice that generates a fluid stream at one end of the flow path, and a laser beam is focused and irradiated on the upstream portion of the orifice in the flow path. A control method of a fine particle sorting device, including a procedure for changing a distance between an objective lens and the objective lens.
(2) A procedure for generating the fluid stream, a procedure for detecting the generated fluid stream, and after these procedures, the relative position of the microchip and the objective lens is changed, and the objective lens is moved to the micro And a method of controlling the microparticle sorting apparatus according to (1) above, including a procedure for moving the chip to a focal position.
(3) The above-described (1) or (2), wherein the distance between the microchip and the objective lens is maintained larger than the working distance of the objective lens with respect to the microchip before the fluid stream is generated. Control method of microparticle sorting apparatus.
(4) After changing the relative position of the microchip and the objective lens, and changing the distance between the microchip and the objective lens to be larger than the working distance, the fluid stream from the orifice The method for controlling a microparticle sorting apparatus according to the above (2) or (3), which includes a procedure for stopping the injection of the microparticles.
(5) The method for controlling a microparticle sorting apparatus according to any one of (2) to (4), wherein the fluid stream is detected by detecting a bright spot in the image of the fluid stream by image recognition. .

In addition, the fine particle sorting apparatus according to the present technology may have the following configuration.
(1) A microchip having a flow path through which a fluid flows and an orifice that generates a fluid stream at one end of the flow path is mounted, and a laser is focused on the upstream portion of the orifice in the flow path. A microparticle sorting apparatus comprising: an objective lens that irradiates; a stream detection unit that detects the fluid stream ejected from the orifice; and a position adjustment unit that changes a distance between the microchip and the objective lens. .
(2) The microparticle sorting apparatus according to (1), wherein the stream detection unit includes a camera that images the fluid stream, and a fluid stream detection light source that irradiates the fluid stream with light.
(3) Fine particle sorting according to (2), further comprising a control unit that detects a bright spot in an image captured by the camera by image recognition, and outputs a signal to the position adjustment unit to adjust the distance. apparatus.
(4) The member according to any one of (1) to (3), wherein a member that prevents movement of the fluid from the orifice toward the objective lens is disposed between the orifice and the objective lens. Microparticle sorting equipment.
(5) The microparticle sorting apparatus according to (4), wherein the member is absorbable with respect to the fluid.

1: Fine particle sorting device (flow cytometer), 11: Chip loading module, 111: Loading section, 112: Chip insertion port, 113: Z-axis stepping motor, 114: Liquid feeding connector section, 115: Sample line, 116 : Sheath / suction line, 12: deflection plate, 13: collection tube holder, 14: objective lens, 15: water droplet blocking plate, 2: microchip, 21: orifice, 3: collection tube, 16: stream detection unit, 161: CCD camera, 162: fluid stream detection light source, 163: Z axis motor, L ₁ , L ₂ : laser, P: cell, S: fluid stream, W: water droplet

Claims (10)

A microchip formed with a flow path through which a fluid flows and an orifice that generates a fluid stream at one end of the flow path;
An objective lens for condensing and irradiating a laser on the upstream portion of the orifice of the flow path;
A method for controlling a microparticle sorting apparatus, including a procedure for changing a distance between the two. Generating the fluid stream;
Detecting the generated fluid stream;
2. The control of the microparticle sorting apparatus according to claim 1, further comprising a step of changing a relative position between the microchip and the objective lens and moving the objective lens to a focal position with respect to the microchip after these procedures. Method.   The method for controlling a microparticle sorting apparatus according to claim 2, wherein a distance between the microchip and the objective lens is maintained larger than a working distance of the objective lens with respect to the microchip before the fluid stream is generated. .   The relative position between the microchip and the objective lens is changed, and the distance between the microchip and the objective lens is changed to be larger than the working distance, and then the fluid stream is ejected from the orifice. The method for controlling a microparticle sorting apparatus according to claim 3, comprising a procedure for stopping.   The method for controlling a microparticle sorting apparatus according to claim 4, wherein the fluid stream is detected by detecting a bright spot in the fluid stream image by image recognition. A microchip having a flow path through which a fluid flows and an orifice that generates a fluid stream at one end of the flow path is mounted.
An objective lens for condensing and irradiating a laser on the upstream portion of the orifice of the flow path;
A stream detector for detecting the fluid stream ejected from the orifice;
A fine particle sorting device comprising: a position adjusting unit that changes a distance between the microchip and the objective lens.   The microparticle sorting apparatus according to claim 6, wherein the stream detection unit includes a camera that images the fluid stream, and a fluid stream detection light source that irradiates the fluid stream with light.   The fine particle sorting device according to claim 7, further comprising a control unit that detects a bright spot in an image captured by the camera by image recognition, and outputs a signal to the position adjustment unit to adjust the distance.   The fine particle sorting device according to claim 8, wherein a member for preventing movement of the fluid from the orifice toward the objective lens is disposed between the orifice and the objective lens.   The microparticle sorting apparatus according to claim 9, wherein the member is absorbable with respect to the fluid.

Images