JP6939063B2 - Flow cytometer - Google Patents

Description

The present invention relates to a droplet detection device and a flow cytometer.

Various methods are known as methods for analyzing fine particles contained in a liquid (see, for example, Patent Documents 1 and 2 below). For example, a flow cytometer is a device that irradiates cells in a liquid with laser light to analyze scattered light and fluorescence. The flow cytometer is provided with a cell sorter that separates and acquires a specific cell group. The cell sorter is a device that separates cells according to the measurement results of cells, and is a liquid that has been separated and flown into a microplate in order to realize efficient drug discovery, minimally invasive diagnosis, tailor-made medical care, etc. in recent years. Applications for inserting drops and cells are increasing (see, for example, Patent Documents 3 to 5 below).

Japanese Unexamined Patent Publication No. 2012-181124 Japanese Unexamined Patent Publication No. 2011-59047 Special Table 2016-521362 JP-A-2015-152439 Special Table No. 10-507525

In such a flow cytometer or cell sorter, a laser light source is used to irradiate minute cells with light. In addition, a photomultiplier tube (PMT) is used to measure scattered light from cells.

In the cell sorter, there is also a method of moving the stage in consideration of the time from the signal of the cell detection unit (photoelectron tube) to the droplet flying and landing on the microplate, but from the nozzle to the microplate Since there is a charged deflection plate on the way and the distance is long, it is unreliable whether the flying droplets landed on the microplate accurately. There is also a risk of foreign matter entering from the air. Further, the cell sorter needs to be manually adjusted while visually observing the origin position of the stage (microplate). It is difficult to visually confirm minute droplets, and when separating a suspension of cells collected from humans, when the operator works with his face close to the microplate, the cells are suspended. The liquid may come into contact with the operator.

An object of the present invention is to provide a droplet detection device and a flow cytometer capable of increasing the reliability and throughput of a cell sorter.

The droplet detection device according to one aspect of the present invention includes a first cylinder portion through which the droplets pass through the internal space and a light emitting portion, and emits light in a direction intersecting the axial direction of the first cylinder portion. It has a light source for the light source and a light receiving unit that is provided at an angle with respect to the optical axis of the light and receives the light refracted by the droplets.

As a result, the light receiving unit is provided at an angle with respect to the optical axis of the light and receives the light refracted by the droplets, so that it is convenient without using a laser light source or a large detector such as a photomultiplier tube. The configuration can detect the passage of droplets. Therefore, the reliability and throughput of the cell sorter to which the droplet detection device is applied can be improved. In addition, reliability means that samples such as precious cells collected from humans and cultured are not wasted due to inaccuracies in position adjustment of cell sorters and microplates, water landing, and detection of landing. Is included.

In the droplet detection device according to one aspect of the present invention, the light emitting unit is a light emitting diode. As a result, the size of the droplet detection device can be reduced, the configuration can be simplified, and the cost can be reduced.

In the droplet detection device according to one aspect of the present invention, the light receiving unit is a photodiode. As a result, the size of the droplet detection device can be reduced, the configuration can be simplified, and the cost can be reduced.

In the droplet detection device according to one aspect of the present invention, further, a second cylinder portion in which the light source is fixed, a lens provided between the light receiving portion and the first cylinder portion, and the lens. Has a third tubular portion that is housed inside. As a result, the light source and the light receiving portion can be arranged in the vicinity of the first cylinder portion.

In the droplet detection device according to one aspect of the present invention, a hole portion is provided on the side portion of the first cylinder portion, and the hole portion is provided coaxially with the optical axis. As a result, it is possible to prevent light from being reflected and diffused on the inner surface of the first cylinder portion to generate unwanted refracted light in the droplet.

In the droplet detection device according to one aspect of the present invention, the first cylinder portion, the second cylinder portion, and a part or all of the third cylinder portion are stereolithographic objects formed by stereolithography. As a result, the second cylinder portion that holds the light source and the third cylinder portion that holds the lens can be manufactured at low cost.

The flow cytometer according to one aspect of the present invention includes the above-mentioned droplet detection device, a droplet generating unit for generating the droplet, a storage unit including a plurality of containers for accommodating the droplet, and the droplet. It has a cell sorter that is arranged between the generation unit and the storage unit and distributes the droplets to each of a plurality of the containers, and the droplet detection device is arranged between the cell sorter and the storage unit. NS.

As a result, the droplet detection device uses a light emitting diode as a light source, and detects the light refracted by the droplet by the photodiode. A flow cytometer requires a laser beam or a photoelectron doubling tube to measure cells in a droplet, but a droplet detector can reliably detect that a droplet has been contained in a compartment. good. Therefore, the passage of droplets can be detected without using a laser light source or a large detector such as a photomultiplier tube. Therefore, it is possible to reduce the size and simplify the configuration of the flow cytometer. Further, since the light emitting diode is used, it is possible to detect the passage of droplets whose flight trajectory is not stable after passing through the cell sorter. Therefore, the reliability and throughput of the cell sorter can be improved.

The flow cytometer according to one aspect of the present invention controls the drive of the motor based on the table on which the storage portion is placed, the motor for driving the table, and the detection signal from the droplet detection device. It is equipped with a control unit. According to this, the motor can operate the table by using the electric signal output by the droplet detection device as a trigger to sequentially move the position of the container for accommodating the droplets. Alternatively, it is also possible to adjust the position of the accommodating portion to the position where the passing signal of the droplet is strongest.

In the flow cytometer according to one aspect of the present invention, the container of the storage portion has an opening, and the inner diameter of the first cylinder portion and the diameter of the opening have the same size. According to this, the droplets that have passed through the first cylinder portion can be reliably stored in the container.

According to the present invention, the reliability and throughput of the cell sorter can be improved.

FIG. 1 is a block diagram showing a configuration example of a flow cytometer according to an embodiment. FIG. 2 is an explanatory diagram for explaining a configuration example of the storage unit. FIG. 3 is a perspective view of the droplet detection device according to the embodiment. FIG. 4 is a top view schematically showing the droplet detection device according to the embodiment. FIG. 5 is a side view schematically showing the droplet detection device according to the embodiment. FIG. 6 is an explanatory diagram for explaining the refraction of light passing through the droplet. FIG. 7 is a graph showing an output voltage waveform of the photodiode according to the first embodiment. FIG. 8 is a graph showing another example of the output voltage waveform of the photodiode according to the first embodiment. FIG. 9 is a graph showing another example of the output voltage waveform of the photodiode according to the first embodiment. FIG. 10 is a graph showing the measurement waveform of the flow cytometer according to the second embodiment. FIG. 11 is a side view schematically showing the droplet detection device according to the first modification. FIG. 12 is a side view schematically showing the droplet detection device according to the second modification. FIG. 13 is a top view schematically showing the droplet detection device according to the third modification. FIG. 14 is a top view schematically showing the droplet detection device according to the fourth modification. FIG. 15 is a top view schematically showing the droplet detection device according to the fifth modification. FIG. 16 is a side view schematically showing the droplet detection device according to the fifth modification.

An embodiment (embodiment) for carrying out the present invention will be described in detail with reference to the drawings. The present invention is not limited to the contents described in the following embodiments. In addition, the components described below include those that can be easily assumed by those skilled in the art and those that are substantially the same. Furthermore, the components described below can be combined as appropriate.

FIG. 1 is a block diagram showing a configuration example of a flow cytometer according to an embodiment. FIG. 2 is an explanatory diagram for explaining a configuration example of the storage unit. As shown in FIG. 1, the flow cytometer 100 includes a droplet generation unit 110, a cell sorter 120, a droplet detection device 1, and a storage unit 130.

The droplet generation unit 110 includes a nozzle 111, a sample supply unit 112, a sheath liquid supply unit 113, and a vibration generating element 114. The sheath liquid supply unit 113 supplies the sheath liquid to the inside of the nozzle 111 via the sheath liquid introduction unit 113a. The sample supply unit 112 supplies the sample to the inside of the nozzle 111 via the sample introduction unit 112a. The sample is a micro object such as a cell. The sheath liquid converges hydrodynamically together with the sample and flows out from the discharge port 111a.

The vibration generating element 114 is, for example, a piezoelectric element or the like. The vibration generating element 114 applies vibration of a predetermined frequency to all or a part of the nozzle 111. As a result, the droplet 101 is discharged from the discharge port 111a. The droplet 101 has a spherical shape in which the circumference of the sample is surrounded by the sheath liquid.

The shape of the droplet 101 is exemplified in Table 1 below, for example. Here, the nozzle diameter d is the diameter of the discharge port 111a. The pressure P is the pressure applied to the inside of the nozzle 111 from the sheath liquid supply unit 113. The flow velocity v is the velocity at which the sheath liquid flows. The frequency f is the frequency of vibration of the vibration generating element 114.

As shown in Table 1, when the nozzle diameter d is 70 μm, the particle diameter D of the droplet 101 is about 138 μm, the particle spacing λ of the droplet 101 is about 356 μm, and the particle mass m of the droplet 101 is about 5.3 μg. be. When the nozzle diameter d is 100 μm, the particle diameter D of the droplet 101 is about 202 μm, the particle spacing λ of the droplet 101 is about 552 μm, and the particle mass m of the droplet 101 is about 18 μg.

The values shown in Table 1 are merely examples, and conditions such as nozzle diameter d, pressure P, flow velocity v, and frequency f can be appropriately changed. The particle size D and the like of the droplet 101 can be appropriately changed according to each of these conditions. In this way, for example, cells having a diameter of about 6 μm are encapsulated inside the droplet 101 having a particle size D of about 200 μm.

As shown in FIG. 1, the cell sorter 120 has a charged portion 121 and a polarizing plate 122. The cell sorter 120 is a device for separating and acquiring the droplet 101 supplied from the droplet generation unit 110 according to the properties of the sample. The charged unit 121 gives a positive or negative charge to the droplet 101 for each droplet 101 based on information such as the size, morphology, and structure of the sample contained in the droplet 101. As a result, the droplet 101 is charged.

A pair of polarizing plates 122 are provided so as to sandwich the flight path of the droplet 101. Voltages having different polarities are applied to one and the other of the polarizing plate 122. The droplet 101 charged by the charged portion 121 passes between the polarizing plates 122. As a result, the flight trajectory of the droplet 101 is changed in a predetermined direction according to the polarity of the droplet 101 and the polarity of the polarizing plate 122.

The droplet detection device 1 is arranged between the cell sorter 120 and the storage unit 130. The droplet detection device 1 passes through the cell sorter 120 and detects the passage of the droplet 101 whose flight trajectory is not stable.

As shown in FIG. 2, the storage unit 130 is a microplate 132 having a plurality of containers 131. The microplate 132 is provided on a stage (not shown). The stage is driven in the first direction Dx and the second direction Dy by the electric motors 134 and 135. Further, the flow cytometer 100 includes a control unit that controls the drive of the electric motors 134 and 135 by utilizing the detection result of the light receiving unit of the droplet detection device 1. The droplet 101 is sorted for each container 131 by the cell sorter 120. In the present embodiment, the droplet detection device 1 is installed directly above the microplate 132. Each of the plurality of containers 131 is provided with an opening, and the inner diameter of the first cylinder portion 51 of the droplet detection device 1 and the diameter of the opening of each container 131 have substantially the same size. As a result, the droplet 101 that has passed through the first cylinder portion 51 can be reliably stored in the container 131. Further, the electric motors 134 and 135 move the microplate 132 based on the detection signal from the droplet detection device 1. As a result, the electric motors 134 and 135 can sequentially move the position of the container 131 for accommodating the droplet 101 by using the detection signal as a trigger. Alternatively, the position of the microplate 132 can be adjusted to the position where the passing signal of the droplet 101 is strongest. When the droplet detection device 1 detects the passage of the droplet 101, the stage is promptly moved to the next position, so that the microplate 132 in which the cells are uniformly contained can be produced.

Next, the configuration of the droplet detection device 1 will be described. FIG. 3 is a perspective view of the droplet detection device according to the embodiment.

As shown in FIG. 3, the droplet detection device 1 includes a light source 10, a first lens 11, a second lens 21, a light receiving unit 54, a first cylinder portion 51, a second cylinder portion 52, and a second. Includes 3 cylinders 53. The first tubular portion 51 is a cylindrical member having an internal space 51a. The diameter of the internal space 51a is, for example, about 7 mm. The droplet 101 from the cell sorter 120 shown in FIG. 1 passes through the internal space 51a of the first tubular portion 51 in the direction along the central axis AX1.

The second tubular portion 52 is a cylindrical member having an internal space 52a. The second tubular portion 52 is connected to the outer periphery of the first tubular portion 51, and the internal space 52a and the internal space 51a communicate with each other. In the present embodiment, the central axis AX2 of the second tubular portion 52 is orthogonal to the central axis AX1 of the first tubular portion 51.

The first lens 11 and the second lens 21 used here are provided with an antireflection coating, and can pass only a predetermined wavelength of the light source 10 and suppress transmission other than that.

The third tubular portion 53 is a cylindrical member having an internal space 53a. The third tubular portion 53 is connected to the outer periphery of the first tubular portion 51 in an inclined state, and the internal space 53a and the internal space 51a communicate with each other. In the present embodiment, the central axis AX3 of the third tubular portion 53 is inclined and intersects with the central axis AX1 of the first tubular portion 51, and is inclined and intersects with the central axis AX2 of the second tubular portion 52.

The light source 10 and the first lens 11 are fixed to the internal space 52a of the second cylinder portion 52. A light emitting diode (LED: Light Emitting Diode) is used as the light source 10. The light source 10 may be any of visible light and infrared light. For example, the wavelength of the light L1 (see FIG. 4) emitted from the light source 10 is 470 nm (blue), 527 nm (green), 587 nm (yellow), 605 nm (orange), 620 nm (red), etc. in visible light. .. The wavelength of infrared light is between 840 nm and 900 nm, for example 870 nm. More preferably, the wavelength is 620 nm or more in the case of visible light because of the spectral sensitivity characteristics of the photodiode 31. Alternatively, in the case of infrared light, a wavelength range of 870 nm or more and 1100 nm or less is more desirable.

Here, as the light receiving unit 54, a module in which the photodiode 31, the amplifier circuit (I / V amplifier), the power supply, and the like are integrated can be used. By integrating the amplifier and the photodiode 31 in close proximity to each other, even weak refracted light can be detected with high accuracy. By using an avalanche photodiode (hereinafter, sometimes referred to as an APD) as the light receiving element and a module with an integrated amplifier, it is possible to measure with a high S / N ratio, and droplets of about φ200 μm. The refracted light of 101 can also be detected. For example, when representing the difference V / W (W is the amount of light), is also a 10 ^5. Of course, in the case of the APD module, the measured light amount is 0.06 μW, but by creating a state in which light other than the light source, such as sunlight, indoor fluorescent lamp, and other UV light, is eliminated, an embodiment described later will be performed. It is possible to measure like this. Further, although the amplifier of the APD module may be affected by the electromagnetic noise of the cell sorter body, the measurement can be performed as an integrated module or by covering the circuit with a lead film or the like.

The first lens 11 is provided between the light source 10 and the first cylinder portion 51. As the first lens 11, for example, a cylinder lens is used. The cylinder lens has an outer diameter shape obtained by cutting out a part of the side surface of the cylinder. The cylinder lens does not have a curved surface when viewed from the central axis AX1 direction of the first cylinder portion 51, but has a curved surface when viewed from a direction intersecting the central axis AX1 and the central axis AX2. With such a configuration, the cylinder lens has a lens action in only one direction. The light L1 emitted from the light source 10 is focused by the first lens 11 and passes across the internal space 51a of the first cylinder portion 51.

The second lens 21 is fixed to the internal space 53a of the third cylinder portion 53. A light receiving portion 54 is provided at the end of the third tubular portion 53. As the second lens 21, for example, a plano-convex lens is used. The light receiving unit 54 includes a photodiode 31. As the photodiode 31, for example, a Si photodiode or an avalanche photodiode is used. When the particle size D of the droplet 101 is, for example, 500 μm, it can be detected by the Si photodiode. When the particle size D of the droplet 101 is, for example, 200 μm, an avalanche photodiode is suitable. The light refracted by the droplet 101 is collected by the second lens 21 and incident on the photodiode 31. As a result, the droplet detection device 1 detects the passage of the droplet 101. Further, in order to prevent the light of the LED light source from being reflected and diffused in the first cylinder portion 51 and deteriorating the detection performance, a hole portion 51b (for which light escapes) is provided on the irradiation destination side of the LED light source of the first cylinder portion 51. (See FIG. 5) is provided. This prevents light from being reflected and diffused on the inner surface (front side of the LED) of the first cylinder portion 51, and undesired refracted light is generated in the droplet 101.

The first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 can be manufactured by using a 3D printer. Stereolithography is the most desirable method for 3D printers. The optical modeling type 3D printer creates a three-dimensional shape model using the three-dimensional shape data created by CAD (Computer Aided Design). In the stereolithography method, a liquid photocurable resin is irradiated with light to form a cured layer, and a plurality of cured layers are laminated to form a stereolithography product. As a result, the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 are integrally formed in the same process. By forming by the stereolithography method, the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 can be formed at low cost. In this case, the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 have a dimensional accuracy of about 0.3 mm because a plurality of layers are laminated. Therefore, the accuracy of the light source 10, the first lens 11, the second lens 21, and the light receiving unit 54 is also limited by the dimensional accuracy of stereolithography. Even in this case, if the droplet 101 has a particle size D of about 200 μm, it can be detected.

Further, by manufacturing using a 3D printer, the light source 10, the first lens 11, the second lens 21, and the light receiving unit 54 can be arranged in the vicinity of each other. Therefore, as shown in FIG. 2, the droplet detection device 1 can be installed directly above the microplate 132. In FIG. 2, the direction in which the container 131 of the microplate 132 opens coincides with the central axis AX1 of the first cylinder portion 51. The opening of the container 131 has, for example, a diameter of about 7 mm (96 wells), but in the case of a smaller diameter, the central axis AX1 may be tilted to provide the first cylinder portion 51. Alternatively, the central axis AX1 of the first tubular portion 51 may be aligned with the trajectory direction of a single line of the droplet 101.

The first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 are preferably painted. The color of the paint is preferably black. As a result, it is possible to prevent light from the outside from entering the internal spaces 51a, 52a, and 53a. Further, the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 are not limited to cylinders. The first tubular portion 51, the second tubular portion 52, and the third tubular portion 53 may have a quadrangular cross-sectional shape, a polygonal shape, an elliptical shape, or a different shape, respectively. Further, the present invention is not limited to the case where the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 are all formed by the stereolithography method. A part of the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 may be formed by a stereolithography method.

Next, a method of detecting the droplet 101 by the droplet detection device 1 will be described. FIG. 4 is a top view schematically showing the droplet detection device according to the embodiment. FIG. 5 is a side view schematically showing the droplet detection device according to the embodiment. FIG. 6 is an explanatory diagram for explaining the refraction of light passing through the droplet. In FIGS. 4 and 5, the second cylinder portion 52 and the third cylinder portion 53 are omitted in order to make the drawings easier to see.

As shown in FIGS. 4 and 5, since the light source 10 is a light emitting diode, the light L1 is non-coherent light and has a spreading angle. The optical axis AX0 of the light L1 is directed in the direction along the first direction Dx. That is, the light L1 is emitted in a direction intersecting the central axis AX1 of the first cylinder portion 51.

In the following description, the first direction Dx, the second direction Dy and the third direction Dz are used. The direction along the central axis AX1 (see FIG. 3) of the first cylinder portion 51 is defined as the third direction Dz. The plane formed by the first direction Dx and the second direction Dy intersects the central axis AX1 (see FIG. 3). The third direction Dz is a direction perpendicular to both the first direction Dx and the second direction Dy. Not limited to this, the second direction Dy may intersect the first direction Dx at an angle other than 90 °. The third direction Dz may intersect the first direction Dx and the second direction Dy at an angle other than 90 °.

As shown in FIG. 4, when viewed from the third direction Dz, the light source 10, the first lens 11, the first cylinder portion 51, the second lens 21, and the photodiode 31 overlap with the optical axis AX0 and are first. Arranged in the direction Dx. The light L1 is focused by the first lens 11. Since the first lens 11 is a cylinder lens, as shown in FIG. 5, the light L2 emitted from the first lens 11 is focused when viewed from the side surface (second direction Dy). On the other hand, as shown in FIG. 4, the light L2 is not focused when viewed from above (third direction Dz). Therefore, the light L2 is applied to the entire surface of the internal space 51a of the first tubular portion 51 through which the droplet 101 passes. By using a cylinder lens as the first lens 11, the droplet 101 can be detected even when the passing position of the droplet 101 varies.

As shown in FIG. 5, the light L2 is divided into a first refracted light L3 that is refracted by the droplet 101 and travels, and a light L4 that travels without being refracted. The second lens 21 and the photodiode 31 are provided at positions inclined with respect to the optical axis AX0 when viewed from the second direction Dy, and can receive the first refracted light L3. In other words, the second lens 21 and the photodiode 31 are arranged at positions offset from the light L4 that is not refracted by the droplet 101. The first refracted light L3 is condensed by the second lens 21 and incident on the photodiode 31. In this way, the photodiode 31 can detect the passage of the droplet 101 by receiving the first refracted light L3.

FIG. 6 shows the relationship between the light L2 incident on the droplet 101 and the first refracted light L3 and the second refracted light L3a. The light L2 is parallel to the reference line HL and is incident at a position shifted upward from the center C of the droplet 101. Here, the reference line HL is a line that intersects the center C of the droplet 101 and is parallel to the first direction Dx (see FIGS. 4 and 5). In other words, the reference line HL is a line parallel to the horizontal direction. The light L2 has an angle θ1 with respect to the normal line NL1 and is incident on the droplet 101. The light L2 is branched into a refracted light L2a and a reflected reflected light L2b at the boundary between the droplet 101 and the air. The refracted light L2a has an angle θ2 with respect to the normal NL1 and travels inside the droplet 101. Further, the reflected light L2b is reflected with an angle θ3 with respect to the normal line NL1. The angle θ1 of the light L2 and the angle θ3 of the reflected light L2b are equal to each other.

Here, the following relationship holds between the angle θ1 of the light L2 and the angle θ2 of the light L2a. However, n1 is the refractive index of the outside of the droplet 101, that is, air, and n1 = 1. n2 is the refractive index of the droplet 101, and n2 = 1.33.

n1 × sinθ1 = n2 × sinθ2

The light L2a traveling inside the droplet 101 is divided into a first refracted light L3 and a reflected light L2c at the boundary between the droplet 101 and air. The first refracted light L3 has an angle θ5 with respect to the normal line NL2 and is emitted to the outside of the droplet 101. Further, the first refracted light L3 has an angle θ8 with respect to the reference line HL. The photodiode 31 shown in FIG. 5 is arranged so that the direction perpendicular to the light receiving surface is inclined with an angle θ8 with respect to the reference line HL. As a result, the photodiode 31 detects the first refracted light L3 that is refracted twice by the droplet 101 and emitted. Further, the reflected light L2c is reflected with an angle θ4 with respect to the normal line NL2. Here, the angle θ4 formed by the light L2a and the normal NL2 and the angle θ5 of the first refracted light L3 have the following relationship.

n1 × sinθ5 = n2 × sinθ4

Similarly, the reflected light L2c traveling inside the droplet 101 is branched into a second refracted light L3a and a reflected light (not shown) at the boundary between the droplet 101 and the air. The second refracted light L3a has an angle θ7 with respect to the normal NL3 and is emitted to the outside of the droplet 101. The photodiode 31 shown in FIG. 5 may be arranged so as to detect the second refracted light L3a. Here, the following relationship holds between the angle θ6 formed by the reflected light L2c and the normal line NL3 and the angle θ7 formed by the second refracted light L3a and the normal line NL3.

n1 × sinθ7 = n2 × sinθ6

In the examples shown in FIGS. 3 to 5, the light L2 incident on the droplet 101 is incident parallel to the reference line HL, but is not limited thereto. The light L2 may enter the droplet 101 at an angle with respect to the reference line HL. In this case as well, the light L2 is repeatedly refracted and reflected, and is emitted to the outside of the droplet 101. For example, as shown in FIG. 6, when the light L2 is incident on the droplet 101 with an angle of θ0 = 14 ° upward with respect to the reference line HL, the first refracted light L3 is with respect to the reference line HL. It is emitted downward at an angle of θ8 = 39 °. In this way, since the angle of the first refracted light L3 can be changed according to the incident angle of the light L2, the first refracted light L3 can be reliably detected by the photodiode 31 shown in FIG.

(First Example)
FIG. 7 is a graph showing an output voltage waveform of the photodiode according to the first embodiment. FIG. 8 is a graph showing another example of the output voltage waveform of the photodiode according to the first embodiment. FIG. 9 is a graph showing another example of the output voltage waveform of the photodiode according to the first embodiment.

Graphs 1 to 3 shown in FIGS. 7 to 9 show the output voltage waveform of the photodiode 31 when glass beads having a diameter of 500 μm are naturally dropped onto the first cylinder portion 51 instead of the droplet 101. .. In each of Graphs 1 to 3, the horizontal axis is time and the vertical axis is output voltage. Graphs 1 to 3 show output voltage waveforms when the falling positions of the glass beads are different when viewed from the third direction Dz.

The wavelength of the light L1 of the light source 10 in this embodiment is 620 nm (red). The first lens 11 uses a biconvex lens. The light receiving sensitivity of the photodiode 31 is 0.4 V / A at a wavelength of 620 nm. The conversion impedance of the amplifier unit connected to the photodiode 31 is 1 × 10 ⁴ V / A.

As shown in Graphs 1 to 3, when the glass beads pass, the photodiode 31 receives the refracted light from the glass beads. As a result, the output voltage becomes large and the passage of the glass beads can be detected. Further, as shown in Graphs 1 to 3, the magnitude of the output voltage may differ depending on the drop position of the glass beads. This showed the possibility of detecting the falling position of the glass beads.

(Second Example)
FIG. 10 is a graph showing the measurement waveform of the flow cytometer according to the second embodiment. As shown in FIG. 1, FIG. 4 shown in FIG. 10 is a measurement waveform when the droplet detection device 1 is applied to the flow cytometer 100. Specifically, as shown in FIG. 1, the droplet detection device 1 is arranged below the cell sorter 120, and detects the droplet 101 passing through the first cylinder portion 51 from between the polarizing plates 122. In Graph 4, the horizontal axis is time and the vertical axis is output voltage.

The nozzle diameter d in this embodiment is 100 μm. Various conditions such as the flow velocity v and the frequency f are as shown in Table 1. The photodiode 31 uses an avalanche diode. The diameter of the light receiving portion of the photodiode 31 is about 3 mm, the maximum incident light quantity (limit) is 0.06 .mu.m, the light receiving sensitivity is -1.5 × ¹⁰ 8 V / W. The wavelength of the light L1 of the light source 10 is 620 nm (red). The voltage value and current value of the light source 10 are adjusted so that a change in the amount of light can be detected without exceeding the maximum amount of incident light of the photodiode 31.

As shown in FIG. 10, the passage of the droplet 101 causes a change in the output voltage. As a result, the flow cytometer 100 of the present embodiment can detect the passage of the droplet 101, which has passed through the cell sorter 120 and whose flight trajectory is not stable, by the droplet detection device 1.

As described above, the droplet detection device 1 of the present embodiment includes the first tubular portion 51 through which the droplet 101 passes through the internal space 51a and the light emitting diode, with respect to the axial direction of the first tubular portion 51. It has a light source 10 that emits light in the intersecting direction, and a photodiode 31 that is provided at an angle with respect to the optical axis AX0 of the light L1 and receives the light (first refracted light L3) refracted by the droplet 101. ..

As a result, a light emitting diode is used as the light source 10, and the first refracted light L3 refracted by the droplet 101 is detected by the photodiode 31. Therefore, the passage of the droplet 101 can be detected without using an expensive and large detector such as a laser light source or a photomultiplier tube. Therefore, the droplet detection device 1 can improve the reliability and throughput of the cell sorter (the microplate 132 in which the cells are contained in all the wells (containers 131) as a finished product). Therefore, the droplet detection device 1 can be miniaturized, and the configuration can be simplified to reduce the cost.

In the droplet detection device 1 according to the present embodiment, the optical axis AX0 of the light L1 is provided so as to be inclined with respect to the horizontal direction. As a result, the direction of the first refracted light L3 refracted by the droplet 101 can be aligned with the position of the photodiode 31.

In the droplet detection device 1 according to the present embodiment, a first lens 11 (cylinder lens) is provided between the light source 10 and the first cylinder portion 51. The light that has passed through the first lens 11 is focused in a direction that intersects the optical axis AX0 and the central axis AX1 of the first cylinder portion 51, that is, when viewed from the second direction Dy, and is focused on the center of the first cylinder portion 51. When viewed from the axis AX1 direction, that is, the third direction Dz, the light is not collected and passes through the first cylinder portion 51. As a result, the entire surface of the internal space 51a of the first tubular portion 51 through which the droplet 101 passes can be irradiated with the light L2. Therefore, the droplet detection device 1 can detect the position where the droplet 101 passes.

The droplet detection device 1 according to the present embodiment further includes a second cylinder portion 52 in which the light source 10 is fixed internally, and a second lens 21 provided between the photodiode 31 and the first cylinder portion 51. The second lens 21 has a third cylinder portion 53 housed therein, and a part or all of the first cylinder portion 51, the second cylinder portion 52, and the third cylinder portion 53 is formed by stereolithography. It is a stereolithography. As a result, the second cylinder portion 52 that holds the light source 10 and the third cylinder portion 53 that holds the second lens 21 can be manufactured at low cost.

The flow cytometer 100 according to the present embodiment includes the above-mentioned droplet detection device 1, a droplet generation unit 110 for generating a droplet 101, and a storage unit 130 including a plurality of containers 131 for accommodating the droplet 101. A cell sorter 120, which is arranged between the droplet generation unit 110 and the storage unit 130 and distributes the droplet 101 to each of a plurality of containers 131, is provided, and the droplet detection device 1 includes the cell sorter 120 and the storage unit 130. Placed in between.

As a result, the droplet detection device 1 uses a light emitting diode as the light source 10 and detects the first refracted light L3 refracted by the droplet 101 by the photodiode 31. Therefore, the passage of droplets can be detected without using an expensive and large detector such as a laser light source or a photomultiplier tube. Therefore, the flow cytometer 100 can be miniaturized, and the configuration can be simplified to reduce the cost. Further, since the light emitting diode is used, it is possible to detect the passage of the droplet 101 whose flight trajectory is not stable after passing through the cell sorter 120.

In the flow cytometer 100 according to the present embodiment, the container 131 of the storage portion 130 has an opening, and the inner diameter of the first cylinder portion 51 and the diameter of the opening have the same size. According to this, the droplet 101 that has passed through the first cylinder portion 51 can be reliably stored in the container 131.

The flow cytometer 100 according to the present embodiment further includes drive units (electric motors 134, 135) that move the storage unit 130 based on the detection signal from the droplet detection device 1. According to this, the electric motors 134 and 135 can sequentially move the position of the container 131 for accommodating the droplet 101 by using the detection signal as a trigger. Alternatively, the position of the accommodating portion 130 can be adjusted to the position where the passing signal of the droplet 101 is strongest.

(First modification)
FIG. 11 is a side view schematically showing the droplet detection device according to the first modification. In the droplet detection device 1A of this modification, the light source 10 and the first lens 11 are arranged so as to be inclined with respect to the reference line HL. In other words, the light L2 inclines upward (Dz side in the third direction) with respect to the reference line HL. The light L2 has an angle θ10 with respect to the reference line HL and is emitted to the droplet 101.

The first refracted light L3 refracted by the droplet 101 is emitted upward with an angle θ11 with respect to the reference line HL. The first refracted light L3 is condensed by the second lens 21 and received by the photodiode 31. Thereby, the passage of the droplet 101 can be detected. In this way, the droplet 101 can be detected even if the light source 10 and the photodiode 31 are arranged so as to be tilted to the same side with respect to the reference line HL.

(Second modification)
FIG. 12 is a side view schematically showing the droplet detection device according to the second modification. In the droplet detection device 1B of this modification, the light source 10 and the first lens 11, the second lens 21 and the photodiode 31 are arranged on the same side with respect to the central axis AX1 of the first cylinder portion 51. The light source 10 and the first lens 11 are arranged so as to be inclined upward (Dz side in the third direction) with respect to the reference line HL. The light L2 has an angle θ12 with respect to the reference line HL and is emitted to the droplet 101.

The second refracted light L3a refracted by the droplet 101 has an angle θ13 with respect to the reference line HL and is emitted downward (opposite the light source 10 and the first lens 11). The second refracted light L3a is condensed by the second lens 21 and received by the photodiode 31. Thereby, the passage of the droplet 101 can be detected. In this way, the droplet 101 can be detected even if the light source 10 and the photodiode 31 are arranged on the same side of the central axis AX1 of the first cylinder portion 51.

(Third modification example)
FIG. 13 is a top view schematically showing the droplet detection device according to the third modification. In the droplet detection device 1C of this modification, the light source 10 and the first lens 11 are arranged at positions overlapping the reference line HL when viewed from the third direction Dz. Further, the second lens 21 and the photodiode 31 are arranged at positions inclined with respect to the reference line HL when viewed from the third direction Dz. In other words, the second lens 21 and the photodiode 31 are arranged at positions that do not overlap with the optical axis of the optical L2. The second lens 21 and the photodiode 31 may be arranged in a plane formed by the first direction Dx and the second direction Dy.

In this case, the first refracted light L3 is refracted by the droplet 101 and emitted from the droplet 101 having an angle θ14 with respect to the reference line HL. The first refracted light L3 is condensed by the second lens 21 and received by the photodiode 31. Thereby, the passage of the droplet 101 can be detected. Further, in this modification, not only the detection of a single droplet 101 but also the passage of a continuous water stream can be detected.

(Fourth modification)
FIG. 14 is a top view schematically showing the droplet detection device according to the fourth modification. In the droplet detection device 1D of this modification, when viewed from the third direction Dz, a plurality of second lenses 21a, 21b, 21c, 21d and a plurality of photographs are provided along the circumferential direction of the first cylinder portion 51. Diodes 31a, 31b, 31c and 31d are provided. In this modification, the plurality of photodiodes 31a, 31b, 31c, and 31d receive the refracted light L31, L32, L33, and L34 emitted from the droplet 101 at different angles, respectively.

The droplet detection device 1D can detect the passing position of the droplet 101 from the light intensity distribution, the time difference, and the like based on the information detected by the plurality of photodiodes 31a, 31b, 31c, and 31d.

(Fifth modification)
FIG. 15 is a top view schematically showing the droplet detection device according to the fifth modification. FIG. 16 is a side view schematically showing the droplet detection device according to the fifth modification. In the droplet detection device 1E of this modification, a biconvex lens is used as the first lens 11A. The light L1 emitted from the light source 10 is focused by the first lens 11A. Since the first lens 11A is a biconvex lens, as shown in FIG. 15, the light L2 emitted from the first lens 11A is focused when viewed from the third direction Dz. Further, as shown in FIG. 16, the light L2 is also focused when viewed from the second direction Dy.

The focal point of the light L2 is formed in the internal space 51a of the first tubular portion 51. The light L2 spreads and travels in the range of the angle θ15 from the focal point. The angle θ15 is, for example, about 40 °. In this modification, the droplet 101 passing through the range of the angle θ15 in the internal space 51a can be detected.

Also in this modified example, as shown in FIG. 15, the second lens 21 and the photodiode 31 are arranged at positions overlapping with the optical axis AX0 when viewed from the third direction Dz. Further, as shown in FIG. 16, the second lens 21 and the photodiode 31 are arranged at positions inclined with respect to the optical axis AX0 when viewed from the second direction Dy. In other words, the second lens 21 and the photodiode 31 are arranged at positions that do not overlap with the light L4 that spreads in a conical shape.

The configurations of the droplet detection devices 1, 1A to 1E and the flow cytometer 100 described above may be appropriately changed. The wavelength of the light L1 from the light source 10 is just an example and can be changed as appropriate. The particle size D and the like of the droplet 101 are merely examples, and are not limited to those shown in Table 1 and the like. Further, the droplet detection devices 1, 1A to 1E are not limited to the case where they are applied to the flow cytometer 100. The droplet detection devices 1, 1A to 1E can also be applied to a dispenser that generates and supplies droplets, or an inkjet nozzle.

1 Droplet detection device 10 Light source 11, 11A First lens 21, 21a, 21b, 21c, 21d Second lens 31, 31a, 31b, 31c, 31d Photo diode 51 First cylinder 51a, 52a, 53a Internal space 52 2 Cylinder 53 3rd Cylinder 54 Light receiving 100 Flow cytometer 101 Droplet 110 Droplet generator 111 Nozzle 111a Discharge port 112 Sample supply 112a Sample introduction 113 Sheath liquid supply 113a Sheath liquid introduction 114 Vibration generating element 120 Cell sorter 121 Charged part 122 Deflection plate 130 Storage part 131 Container AX1, AX2, AX3 Central axis d Nozzle diameter NL1, NL2, NL3 Normal line

Claims (7)

A light source that includes a first cylinder portion through which droplets pass through the internal space and a light emitting portion and emits light in a direction intersecting the axial direction of the first cylinder portion, and a light source that is tilted with respect to the optical axis of the light. A droplet detection device provided with a light receiving unit for receiving light refracted by the droplets.
A droplet generating unit that generates the droplets and
A storage unit including a plurality of containers for storing the droplets,
It has a cell sorter which is arranged between the droplet generation unit and the storage unit and which distributes the droplets into each of a plurality of the containers.
The droplet detection device is arranged between the cell sorter and the storage portion, and is arranged.
The container in the compartment has an opening and
The inner diameter of the first cylinder and the diameter of the opening have the same size.
Flow cytometer. The flow cytometer according to claim 1, wherein the light emitting unit is a light emitting diode. The flow cytometer according to claim 1 or 2, wherein the light receiving portion is a photodiode. Further, it has a second tubular portion in which the light source is fixed, a lens provided between the light receiving portion and the first tubular portion, and a third tubular portion in which the lens is housed. The flow cytometer according to any one of claims 1 to 3. A hole is provided on the side of the first cylinder.
The flow cytometer according to any one of claims 1 to 4, wherein the hole is provided coaxially with the optical axis. The flow cytometer according to claim 4, wherein the first cylinder portion, the second cylinder portion, and a part or all of the third cylinder portion are stereolithographic objects formed by stereolithography. The table on which the storage unit is placed and
The motor that drives the table and
The flow cytometer according to any one of claims 1 to 6, further comprising a control unit that controls drive of the motor based on a detection signal from the droplet detection device.

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