JP6885052B2 - Particle counting device and particle counting method - Google Patents

Description

The present invention relates to a particle counting device and a particle counting method for counting particles contained in ejected droplets.

In recent years, with the progress of stem cell technology, the development of a technique for forming a tissue by ejecting a plurality of cells by inkjet has been carried out. When ejecting a droplet containing a particulate matter typified by a cell, it is important to receive light on how many particulate matter is contained in the ejected droplet.

As an example of an apparatus having such a function, a discharge apparatus that receives light on the number of granules contained in the liquid material has been proposed (see, for example, Patent Document 1). In this discharge device, the number of particles contained in the liquid material passing through the detection unit provided between the cavity of the discharge device and the nozzle can be detected.

An object of the present invention is to provide a particle counting device capable of improving the counting accuracy of particles contained in the discharged droplets.

The particle counting device of the present invention as a means for solving the above problems includes a droplet ejection means for ejecting particles containing particles capable of emitting light when irradiated with light, and a droplet ejection means for ejecting the particles. Based on the light irradiation means for irradiating the droplets with the light, two or more light receiving means for receiving light emission from the particles irradiated with the light, and the light emission received by the two or more light receiving means. It has a particle counting means for counting the particles contained in the droplets.

According to the present invention, it is possible to provide a particle counting device capable of improving the counting accuracy of particles contained in the discharged droplets.

FIG. 1 is an explanatory diagram showing an example of the particle counting device of the present invention. FIG. 2 is an explanatory diagram showing an example of the positional relationship between the two light receiving means in the particle counting device shown in FIG. FIG. 3 is an explanatory diagram of an image of light emission obtained by the two light receiving means having the positional relationship shown in FIG. FIG. 4A is an explanatory diagram showing an example of a simulation image of the luminance value of the emission and the shape of the light emitting surface on the light receiving surface when the two emissions overlap. FIG. 4B is an explanatory diagram showing another example of a simulation image of the luminance value of the emission and the shape of the light emitting surface on the light receiving surface when the two emissions overlap. FIG. 4C is an explanatory diagram showing another example of a simulation image of the luminance value of the emission and the shape of the light emitting surface on the light receiving surface when the two emissions overlap. FIG. 5A is an explanatory diagram showing an example of a simulation image of the brightness value of light emission and the shape on the light receiving surface of light emission when the particles are present in the vicinity of the outer edge portion of the droplet. FIG. 5B is an explanatory diagram showing another example of a simulation image of the brightness value of light emission and the shape of the light emitting surface on the light receiving surface when the particles are present in the vicinity of the outer edge portion of the droplet. FIG. 6A is an image showing an example of the luminance value of the light emitted by the light receiving means and the shape of the light emitting surface on the light receiving surface. FIG. 6B is an image showing another example of the luminance value of the light emitted by the light receiving means and the shape of the light emitting surface on the light receiving surface. FIG. 6C is an image showing another example of the luminance value of the light emitted by the light receiving means and the shape of the light emitting surface on the light receiving surface. FIG. 7 is a graph showing an example of a calibration curve for determining the number of particles based on the luminance value of the light emitted by two or more light receiving means. FIG. 8 is a flowchart showing an example of a flow for counting particles contained in the droplets ejected from the droplet ejection means using the particle counting apparatus of the present invention. FIG. 9 is a flowchart showing another example of the flow of counting the particles contained in the droplets ejected from the droplet ejection means using the particle counting apparatus of the present invention. FIG. 10 is a flowchart showing another example of the flow of counting the particles contained in the droplets ejected from the droplet ejection means using the particle counting apparatus of the present invention. FIG. 11 is a flowchart showing another example of the flow of counting the particles contained in the droplets ejected from the droplet ejection means using the particle counting apparatus of the present invention. FIG. 12 is a flowchart showing another example of the flow of counting the particles contained in the droplets ejected from the droplet ejection means using the particle counting apparatus of the present invention. FIG. 13 is a flowchart showing another example of the flow of counting the particles contained in the droplets ejected from the droplet ejection means using the particle counting apparatus of the present invention. FIG. 14 is an explanatory diagram showing another example of the particle counting device of the present invention. FIG. 15 is an explanatory diagram showing still another example of the particle counting device of the present invention. FIG. 16 is a flowchart showing an example of a flow in which the number of particles contained in the droplets landing on the adherend is set to a predetermined number by using the particle counting device of the present invention. FIG. 17 is a flowchart showing another example of a flow in which the number of particles contained in the droplets landing on the adherend is set to a predetermined number by using the particle counting device of the present invention.

(Particle counting device and particle counting method)
The particle counting device of the present invention includes a droplet ejection means for ejecting droplets containing particles capable of emitting light when irradiated with light, and a light irradiation means for irradiating the droplets ejected from the droplet ejection means with light. It has two or more light receiving means for receiving light emitted from particles irradiated with light, and a particle counting means for counting particles contained in the droplet based on the light emitted by the two or more light receiving means. Further, if necessary, other means are provided.
Further, it is preferable that each means in the particle counting device of the present invention is communicably connected and the droplet ejection means is controlled based on the number of particles counted by the particle counting means. Further, the particle counting device of the present invention preferably has a feedback unit that controls the droplet ejection means based on the result of counting the particles contained in the droplets landing on the adherend.

The particle counting method of the present invention includes a droplet ejection step of ejecting droplets containing particles capable of emitting light when irradiated with light, a light irradiation step of irradiating the ejected droplets with light, and irradiation of light. It includes a light receiving step of receiving the light emitted from the particles by two or more light receiving means and a particle counting step of counting the particles contained in the droplet based on the light received by the two or more light receiving means, and further necessary. Other steps are included accordingly.
Further, the particle counting method of the present invention preferably includes a first feedback step of controlling the ejection of droplets in the droplet ejection step based on the number of particles counted in the particle counting step. Further, the particle counting method of the present invention may include a second feedback step of controlling the ejection of the droplet in the droplet ejection step based on the result of counting the particles contained in the droplet landing on the adherend. preferable.

The particle counting method of the present invention can be preferably performed by the particle counting device of the present invention.
Hereinafter, the details of the particle counting method of the present invention will be clarified through the description of the particle counting device of the present invention.

In the ejection device of Patent Document 1, the particle counting device of the present invention counts the particles contained in the droplet before ejecting the droplet, that is, before reaching the opening of the ejection nozzle. It is based on the finding that the number of particles contained in the droplet does not always match and the counting accuracy of the particles decreases.

Further, when counting the particles contained in the discharged droplets, when the particles are irradiated with light to emit light and the particles are counted based on the light emission from the particles, as in the particle counting device of the present invention. , There are the following problems.
The ejected droplets become spherical or ellipsoidal due to atmospheric pressure or the like, and have a depth in the direction of receiving the particles, so that the particles tend to overlap. Therefore, there is a problem that it becomes difficult for the particles to receive light when the light emission from the particles overlaps. In addition, since there is a depth in the direction in which the particles are received, the brightness value of the light emission from the particles existing on the back side of the light receiving means is attenuated by refraction or scattering as much as it passes through the droplets. There is a problem that the brightness value of light emission tends to vary depending on the position in the droplet with respect to the light receiving means. Further, when the particles are present near the outer edge of the droplet, when the particles are irradiated with light to emit light, there is a problem that the light emitted from the particles is reflected inside the spherical surface of the droplet and is easily blurred.
When counting the particles contained in the ejected droplets, the particle counting device of the present invention makes it difficult to clearly draw a calibration line based on the brightness value of light emission due to these problems, and determines the number of particles. This is based on the findings found by the present inventors that it becomes difficult to count particles and the counting accuracy of particles decreases.

<Drop ejection means and droplet ejection process>
The droplet ejection means is a means for ejecting droplets containing particles that can emit light when irradiated with light.
The droplet ejection step is a step of ejecting droplets containing particles capable of emitting light when irradiated with light, and can be preferably performed by the droplet ejection means.

The droplet ejection means is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a piezoelectric pressurizing method using a piezoelectric element, a thermal method using a heater, or an electrostatic attraction method is used to induce a liquid. Examples thereof include an inkjet head of an electrostatic method and a membrane vibration method using a piezoelectric element. Among these, a membrane vibration type inkjet head is preferable.
The membrane vibration type inkjet head is a method of ejecting droplets by the inertial force due to vibration, and the upper part of the inkjet head can be released to the atmosphere. Therefore, especially when the particles are cells, the heat and electric field on the cells are generated. , Damage such as pressure can be reduced.
The piezoelectric element used in the membrane vibration type inkjet head is not particularly limited and may be appropriately selected depending on the intended purpose. For example, an element using lead zirconate titanate (PZT) is preferable.

The particles capable of emitting light are not particularly limited and may be appropriately selected depending on the intended purpose. For example, cells expressing a fluorescent protein, stained cells stained with a fluorescent dye, inorganic fine particles stained with a fluorescent dye, and the like. Examples thereof include organic polymer particles dyed with a fluorescent dye.

The organic polymer particles dyed with the fluorescent dye are not particularly limited and may be appropriately selected depending on the intended purpose. For example, SPHERO Fluorescent Nile Red parts (manufactured by Bay Bioscience Co., Ltd., 1% (w / v)) , Diameter 10 μm to 14 μm) and the like.
The fluorescent protein is not particularly limited and may be appropriately selected depending on the intended purpose. For example, green fluorescent protein (GFP; Green Fluorescent Protein), red fluorescent protein (RFP; Red Fluorescent Protein), and yellow fluorescent protein (YFP). ; Yellow Fluorescent Protein) and the like.
The fluorescent dye in the stained cells is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include cell tracker orange and cell tracker red.

When the particles are agglomerated, the concentration of the particles in the suspension and the number of particles in the suspension can be adjusted by adjusting the concentration of the particles in the suspension containing the particles to be filled in the droplet ejection means. From the theory that follows the Poisson distribution, the number of particles in the suspension can be adjusted to a few or less.
The liquid component of the suspension is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ion-exchanged water.
The diameter of the droplet is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 25 μm or more and 150 μm or less. When the diameter of the droplet is 25 μm or more, the diameter of the droplet is not small, the diameter of the contained particles is small, and the types of applicable particles are not reduced. If it is 150 μm or less, there is no problem as a droplet, and in order to eject the droplet, it is not necessary to increase the hole diameter of the inkjet head, and the ejection of the droplet is unlikely to become unstable.
Further, assuming that the diameter of the droplet is R and the diameter of the particle is r, it is preferable that R> 3r. When R> 3r, since the diameter of the particles is not large with respect to the diameter of the droplets, it is less likely to be affected by the edges of the droplets, and the counting accuracy of the particles is less likely to decrease.

<Light irradiation means and light irradiation process>
The light irradiation means is a means for irradiating the droplets ejected from the droplet ejection means with light.
The light irradiation step is a step of irradiating the discharged droplets with light, and can be preferably performed by the light irradiation means.

As the light irradiation means, it is preferable that light can be irradiated in synchronization with the ejection of droplets by the droplet ejection means. As a result, the droplets ejected from the droplet ejection means can be more reliably irradiated with light.
Here, "synchronizing" means that the light irradiating means irradiates the light when the droplets are ejected and reach a predetermined position. That is, the light irradiating means irradiates the light with a delay of a predetermined time with respect to the ejection of the droplet by the droplet ejection means.

The light irradiation means is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a solid-state laser, a semiconductor laser, and a dye laser.
Examples of the solid-state laser include a YAG laser, a ruby laser, and a glass laser.
Examples of commercially available YAG lasers include Explorer ONE-532-200-KE (manufactured by Spectra Physics Co., Ltd.).
Among these, it is preferable that pulsed light can be irradiated by pulse oscillation.

The pulse width of the pulsed light is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 μs or less, and more preferably 1 μs or less.
The energy per unit pulse is not particularly limited and can be appropriately selected according to the purpose, and largely depends on the optical system such as the presence or absence of light collection, but 0.1 μJ or more is preferable, and 1 μJ or more is more preferable. ..

<Light receiving means and light receiving process>
The light receiving means is a means for receiving light emitted from the particles irradiated with light.
The light receiving step is a step of receiving light emitted from the particles irradiated with light by two or more light receiving means, and can be preferably performed by the light receiving means.

The particle counting device of the present invention has two or more light receiving means. If the particle counting device has two or more light receiving means, even if one light receiving means receives the overlapping state, the other light receiving means can receive the non-overlapping state. , Particles contained in the droplet can be accurately counted based on the light emitted by other light receiving means.

The light emitted from the irradiated particles is emitted from the particles in all directions. Therefore, the two or more light receiving means are not particularly limited as long as they are arranged at positions where light can be received, and can be appropriately selected according to the purpose, but the angle formed by each light receiving direction is 0 °. It is preferable to arrange it in a position where it does not become. It is advantageous in that information on a state in which there is little overlap of light emission can be obtained.

It is preferable that at least one of the two or more light receiving means is arranged so that the light receiving direction thereof is located substantially orthogonal to the light receiving direction of the other light receiving means. As a result, when one light receiving means and another light receiving means are used, there is little overlap of light emission when selecting any of the information received by the one light receiving means and the other light receiving means. You can select information. In addition, substantially orthogonal means 80 ° or more and 100 ° or less.

The light receiving direction in the light receiving means other than the above 2 light receiving means is not particularly limited and may be appropriately selected depending on the intended purpose. When there are two or more light receiving means, when two or more light receiving means are arranged so as to be located on the same plane, the angle formed by the light receiving directions of adjacent light receiving means is equally divided by the number of light receiving means at 360 °. It is preferable to make the angle. For example, when the three light receiving means are arranged so as to be located on the same plane, it is preferable that the three light receiving means are arranged so that the light receiving directions of the adjacent light receiving means are respectively 120 °.

The light receiving means is preferably arranged so that the light receiving direction is located substantially orthogonal to the droplet ejection direction, and all the light receiving means are located in the light receiving direction substantially orthogonal to the droplet ejection direction. It is more preferable that they are arranged in such a manner. This makes it easy to adjust the position of the light receiving means, which is advantageous in that the structure of the particle counting device is not complicated.
In order to improve the contrast, it is preferable to arrange the light receiving means at a position where the light emitted from the light irradiating means is not directly incident.

As the light receiving means, it is preferable to obtain information on the brightness value of the light emitted in the substantially normal direction of the light receiving surface and the shape of the light emitted on the light receiving surface. As a result, the particle counting means counts the particles contained in the droplet based on the brightness value of the light emission, and counts the particles contained in the droplet based on the shape information on the light receiving surface of the light emission. Since at least one of the second counting processes for counting can be performed, the counting accuracy of particles can be improved.

As the light receiving means, it is preferable that light emission can be received in synchronization with the ejection of droplets by the droplet ejection means. As a result, the droplets ejected from the droplet ejection means are irradiated with light from the light irradiation means, and the light emitted from the particles can be received more reliably.
Here, "synchronization" means that, for example, when the droplets are ejected and reach a predetermined position, the droplets are irradiated with light, and the light receiving means receives the light emission at the timing when the particles emit light. .. That is, the light receiving means detects the light emission with a delay of a predetermined time with respect to the ejection of the droplet by the droplet ejection means and the irradiation of light by the light irradiation means.

The light receiving means is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a camera having a one-dimensional element, a two-dimensional element, and the like. Among these, a camera having a two-dimensional element is preferable. When the light receiving means is a camera having a two-dimensional element, it is advantageous in that not only the brightness value of the light emission but also the shape of the light receiving surface of the light emission can be easily obtained.

Examples of the one-dimensional element include a photodiode and a photosensor. Among these, photomultiplier tubes and avalanche photodiodes are preferable. If the one-dimensional element is a photomultiplier tube or an avalanche photodiode, highly sensitive measurement is possible.
Examples of the two-dimensional element include a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor) image pickup device, a gate CCD, and the like.
As the light receiving means, a camera having a CMOS image sensor is preferable.
As a commercially available camera having a CMOS image sensor, a high-sensitivity camera (pco.edge, sCMOS, manufactured by Tokyo Instruments Co., Ltd.) is preferable.

If the light emitted from the particles is weaker than the light emitted by the light irradiating means, a filter that attenuates the wavelength range of the light may be installed on the light receiving surface side of the light receiving means. By installing the filter, the light receiving means can receive light emission with less noise.
Examples of the filter include a notch filter that attenuates a specific wavelength region including a wavelength of light.

As described above, the light emitted from the light irradiating means is preferably pulsed light, but may be continuously oscillated light. In this case, it is preferable to control the light receiving means so that the light emission can be received at the timing when the continuously oscillated light is irradiated to the ejected flying droplets.

<Particle counting means and particle counting process>
The particle counting means is a means for counting particles contained in a droplet based on the light emitted by two or more light receiving means.
The particle counting step is a step of counting the particles contained in the droplet based on the light emitted by two or more light receiving means, and can be preferably performed by the particle counting means.

The particle counting means has a first counting process for counting the particles contained in the droplet based on the brightness value of the light emission, and a second counting process for counting the particles contained in the droplet based on the shape information on the light receiving surface of the light emission. It is preferable to perform at least one of the two counting processes.
Next, the first counting process and the second counting process performed by the particle counting means will be described.

[First counting process for counting particles based on the brightness value of light emission]
The first counting process for counting particles based on the brightness value of light emission is not particularly limited and may be appropriately selected depending on the intended purpose. For example, (1) light emission received by at least two light receiving means. Examples include a process of determining the number of particles using a calibration curve acquired in advance based on a luminance value, and (2) a process of counting the number of light emission that is the largest among the number of light emitted by the light receiving means as the number of particles. Be done.

In the process (1) above, as long as the particles are capable of transmitting light emission, the number of particles can be determined using a calibration curve based on the total brightness value of the light emission even when the light emission overlaps. Even for particles that do not transmit light, in order to obtain the brightness value of light emission from two or more light receiving means, it is possible to count the particles based on the brightness value of light emission that does not overlap or has little overlap of light emission. it can.

In the process (2) above, the number of light emission whose brightness value is within a predetermined range is obtained based on the brightness value of the light emission received by each light receiving means, and the largest number of light emission among the obtained number of light emission is obtained. The number can be counted as the number of particles.

Among these, the treatment of (1) above is preferable. If the first counting process is the process (1) above, it is advantageous in that the accuracy of counting the number of particles is improved.
When performing the first counting process, a one-dimensional element may be used or a two-dimensional element may be used as the light receiving means.

[Second counting process for counting particles based on shape information on the light emitting surface]
The second counting process for counting particles based on the shape information on the light receiving surface of light emission is not particularly limited and may be appropriately selected depending on the intended purpose. For example, (3) the shape on the light receiving surface of light emission. The number of centers of the radius of curvature within a predetermined range of the obtained radius of curvature is counted as the number of particles. (4) The length of the outer circumference of the shape on the light receiving surface of light emission is predetermined. Processing to count the number of light emission within the range, (5) Processing to count the number of inflection points on the outer circumference of the shape on the light receiving surface as the number of particles, (6) Processing to count the number of curvatures on the outer circumference of the shape on the light receiving surface of light emission Examples include counting the number of times the sign of the inclination of the tangent changes as the number of particles.

In the process (3) above, when the radius of curvature is smaller than the predetermined range, refracted light or scattered light other than the light emitted from the particles is received, so light emission having the radius of curvature smaller than the predetermined range is excluded. To do. In addition, when the radius of curvature is larger than a predetermined range, when the particles are present near the outer edge of the droplet, when the particles are irradiated with light to emit light, the light emitted from the particles is inside the spherical surface of the droplet. Since the outer edge of the droplet appears to emit light due to the reflection phenomenon, it is judged that the emission is not from the particles and is excluded. The number of centers of the circle when the radius of curvature within a predetermined range is calculated out of the radius of curvature calculated based on the shape information on the light receiving surface of light emission can be counted as the number of particles.

The process (4) above replaces the radius of curvature in the process (3) with the length of the outer circumference of the shape on the light receiving surface of light emission.

In the process of (5) above, for example, when two light emissiones overlap, the number of inflection points becomes two, and when three light emissiones overlap, the number of inflection points becomes three. Can be counted as the number of particles.

In the process (6) above, for example, when two emissions overlap, the sign of the slope of the tangent changes twice, and when three emissions overlap, the sign of the inclination of the tangent changes three times. Can be counted as the number of particles.

Among these, the treatment of (3) above is preferable. That is, the particle counting means calculates the radius of curvature based on the information of the shape on the light receiving surface of light emission, and counts the number of centers of the circle when the radius of curvature within a predetermined range is calculated as the number of particles. It is more preferable to carry out the counting process of. When the second counting process is the process of (3) above, it is advantageous in that counting is easy even if there are a plurality of particles, and the accuracy of counting the particles is improved.

When performing the second counting process, it is preferable to use a two-dimensional element as the light receiving means. If the light receiving means is a two-dimensional element, it is advantageous in that a two-dimensional image can be easily obtained.
Based on the two-dimensional image obtained from the light receiving means, software that performs image processing for calculating the radius of curvature based on the shape of the light emitting surface on the light receiving surface may be used.
Examples of software that performs image processing include ImageJ (manufactured by the National Institutes of Health, open source).

[Order of first counting process and second counting process]
The order of the first counting process and the second counting process is not particularly limited and may be appropriately selected depending on the purpose, but the particle counting means performs the first counting process and cannot count the particles. When it is determined, it is preferable to perform a second counting process to count the particles.
Since the processing speed of the first counting process is faster than that of the second counting process, the first counting process is performed first. During the first counting process, for example, because the light emitted from the particles is reflected inside the spherical surface of the droplet, it may be difficult to count the particles by a calibration curve based on the brightness value of the light emission. In this case, by providing a non-determinable region on the calibration curve in advance, it is determined that the particles cannot be counted by the first counting process, and by switching to the second counting process, the counting accuracy of the particles contained in the droplets is increased. Can be improved.

[First feedback process by particle counting means]
In the first feedback step, when it is desired to reduce the number of particles contained in the droplet landed in the well as an adherend to one, the particle counting means uses zero particles in the flying droplet. When it is determined that there is, it is preferable to control the droplet ejection means to further eject the droplet. Further, it is more preferable to prevent the flying droplets, which are determined to have a number of particles other than one, from adhering to the adherend. As a result, it is possible to prevent the adherend from being soiled. Further, after the flying droplets determined to have one or more particles land on the adherend, the next droplet is landed at a position different from the position where the droplets landed on the adherend. You may want to move to.

Further, in the first feedback step, when it is desired to set the number of particles contained in the droplets to be landed in the well to a predetermined number, the particle counting means means that the number of particles contained in the flying droplets is less than the predetermined number. When it is determined that there is, it is preferable to control the droplet ejection means to further eject the droplets. Further, when the particle counting means determines that the number of particles contained in the flying droplets is 0 or a predetermined number or more, it is preferable to prevent the flying droplets from landing on the adherend. In this regard, the step of counting the particles contained in the flying droplets and feeding them back to the droplet ejection means is a process of counting the particles contained in the droplets landing on the adherend and feeding them back to the droplet ejection means, which will be described later. It is more productive than the process.
After ejecting the droplets a predetermined number of times by the droplet ejection means, the brightness value of the emission and the shape information on the light receiving surface of the emission stored in the storage means described later are read out and included in each of the ejected droplets. The particles to be generated may be counted.

The particle counting means includes a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), a main memory, etc. that control each operation of the particle counting device of the present invention, and includes the entire particle counting device. Various processes are executed based on the control program for controlling the operation.

<Feedback unit>
The feedback unit can count the particles contained in the droplets that have landed on the adherend, and can feedback-control the droplet ejection means based on the number of the counted particles.
The feedback unit includes an imaging means and a particle counting means.
Hereinafter, in order to distinguish between the particle counting means of the feedback unit and the particle counting means of the particle counting device of the present invention, the particle counting means of the above-mentioned particle counting device is referred to as a "first particle counting means", and the feedback unit is used. The particle counting means may be referred to as a "second particle counting means".

The image pickup means includes a light irradiation means, a dichroic mirror, an objective lens, an image pickup device, and a scanning unit.
Hereinafter, in order to distinguish between the light irradiation means of the feedback unit and the light irradiation means of the particle counting device of the present invention, the light irradiation means of the particle counting device described above is referred to as a "first light irradiation means", and the feedback unit The light irradiation means may be referred to as a "second light irradiation means".

An objective lens is installed in the imaging means at a position where the landed droplets can be observed. The laser beam emitted from the second light irradiation means of the imaging means passes through the objective lens through the dichroic mirror and irradiates the fluorescent particles in the well as an adherend. Image information can be obtained by passing the fluorescence emitted from the fluorescent particles through the objective lens and incident on the image sensor.

Examples of the second light irradiation means include a solid-state laser, a semiconductor laser, a dye laser, and an LED. When the fluorescent particles existing in the droplets landed in the wells are irradiated with the laser light of the light irradiation means, the fluorescent particles emit light, so that the counting accuracy of the particles is improved.

The objective lens is not particularly limited and can be appropriately selected according to the purpose. However, if a high-magnification lens is used, the counting accuracy is improved, but the field of view is narrowed, so that the droplets land outside the field of view. It becomes easy, and the count error frequency tends to be high. On the other hand, if a low-magnification lens is used, the field of view is widened, so that the frequency of count errors can be reduced, but it becomes difficult to capture cells of 10 μm or less.
When capturing cells of 10 μm or less with an objective lens, it is preferable to use an objective lens with a field of view of about 0.5 mm square from the viewpoint of the landing position accuracy of the droplet ejection means. Examples of commercially available products include an infinite correction objective lens M-PLAN APO 5X (manufactured by Mitutoyo Co., Ltd.).

The image pickup device is not particularly limited and may be appropriately selected depending on the intended purpose. In the case of acquiring an image of particles, for example, a two-dimensional element such as a CCD, CMOS, or gate CCD can be used. Further, when determining the number of particles based on the brightness value of light emission, a one-dimensional element such as a photodiode or a photosensor may be used as the image sensor. When a high-sensitivity image sensor is required, it is preferable to use a photomultiplier tube, an avalanche photodiode, or the like.
When the second light irradiation means is not used, the fluorescent particles in the droplet landing on the adherend are hidden in the shadow of the peripheral portion of the droplet, which makes it difficult to count the particles as shown in FIG. 5B. .. For this reason, it is preferable to provide a time delay so that an image is taken after the droplet has landed and before it dries. When the second light irradiation means is used, the fluorescent particles in the droplet emit fluorescence, which facilitates confirmation of the fluorescent particles, so that it is possible to take an image before the droplet dries.
Since the fluorescence emitted by the fluorescent particles is weaker than that of the laser light emitted by the second light irradiation means, a filter for attenuating the wavelength range of the laser light may be installed in the front stage (light receiving surface side) of the image pickup element. This makes it easier to obtain an image of fluorescent particles having extremely high contrast in the image sensor. Examples of the filter include a notch filter that attenuates a specific wavelength region including the wavelength of laser light.

The scanning unit is for moving the focal position of the objective lens to scan and image fluorescent particles. If the focal position with respect to the fluorescent particles shifts, the contrast of the image deteriorates, and it tends to be difficult to accurately count the number of particles from the acquired image. Therefore, after the droplet has landed in the well, a plurality of images are acquired by the scanning unit, so that an image having high contrast can be selected and acquired.

The feedback unit can count the particles contained in the droplets landing on the adherend, and compares the obtained count value with the count value obtained by the first particle counting means. It can be fed back to the droplet ejection means.
As a method for counting the particles contained in the droplets that have landed on the adherend, the above-mentioned methods such as the first counting process and the second counting process can be applied.

When a two-dimensional element is used as the image sensor, the feedback unit calculates the area value of the part where the fluorescent particles are emitting light by image processing and compares it with a preset threshold value to obtain the particles contained in the droplets. Can be counted. In this case, the feedback unit preferably has a number determination algorithm based on the shape of the image.
Further, when a one-dimensional element is used as the image pickup device, the feedback unit can count the particles contained in the droplet by comparing with a preset threshold value based on the obtained luminance value of light emission. ..

The particle counting device of the present invention is not limited to counting the particles contained in the flying droplets, and uses a feedback unit without counting the particles contained in the flying droplets. , The particles contained in the droplet after landing on the adherend may be counted.

[Second feedback step by the second particle counting means]
In the second feedback step, when it is desired to reduce the number of particles contained in the droplet landed in the well as an adherend to one, the second particle counting means is the number of particles contained in the landed droplet. When it is determined that the number of particles is 0, it is preferable to control the droplet ejection means to further eject the droplets. Further, when the second particle counting means determines that the number of particles contained in the landed droplet is one or more, it shifts to the next step such as landing at a position different from the landed position. It may be.

Further, in the second feedback step, when it is desired to set the number of particles contained in the landed droplet to a predetermined number, the second particle counting means uses the second particle counting means to reduce the number of particles contained in the landed droplet to a predetermined number. When it is determined that the above is true, it is preferable to control the droplet ejection means to further eject the droplets. Further, when the second particle counting means determines that the number of particles contained in the landed droplet exceeds a predetermined number, the next droplet is landed at a position different from the position where the landed object is landed. You may move to the next process such as.
After ejecting the droplets a predetermined number of times from the droplet ejection means, the brightness value of the emission and the shape information on the light receiving surface of the emission stored in the storage means described later are read out and included in each of the ejected droplets. The particles to be generated may be counted.

As described above, the feedback step is a step in which the first or second particle counting means feeds back to the droplet ejection means, and can be preferably performed by the feedback unit.

<Other means and other processes>
The other means are not particularly limited and may be appropriately selected depending on the intended purpose, but preferably include an optical system, a driving means, a storage means, and an object to be adhered.
Other steps are preferably performed by other means.

The optical system is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a lens for condensing light emitted from a light irradiating means into droplets and a light receiving means for filtering light are used. Examples thereof include a filter for facilitating the reception of light emission.

The drive means is not particularly limited and may be appropriately selected depending on the intended purpose. For example, when the droplet discharge means is an inkjet head by a piezoelectric pressurization method, a means for inputting a drive voltage to the droplet discharge means. And so on. In this case, the driving means can deform the piezoelectric element to eject minute droplets.

The storage means is not particularly limited as long as it can store the luminance value of the light emitted by the light receiving means and the shape information of the light emitting surface on the light receiving surface, and can be appropriately selected according to the purpose. Examples thereof include RAM. ..

The adherend to which the ejected droplets adhere is not particularly limited and may be appropriately selected. For example, when the particles are cells, wells and the like can be mentioned.

Here, an example of the particle counting device in the present invention will be described with reference to the drawings.
In each drawing, the same components may be designated by the same reference numerals, and duplicate description may be omitted. Further, the number, position, shape, etc. of the following constituent members are not limited to the present embodiment, and can be a preferable number, position, shape, etc. for carrying out the present invention.

FIG. 1 is an explanatory diagram showing an example of the particle counting device of the present invention.
As shown in FIG. 1, the particle counting device 100 includes a droplet discharging means 10, a driving means 20, a light irradiating means 30, light receiving means 41 and 42, and a particle counting means 51.

The droplet ejection means 10 accommodates a fluorescent particle suspension 200 containing fluorescent particles 201 in the cavity 11, and a spherical or ellipsoid containing the fluorescent particles 201 is formed by deformation of a piezoelectric element (not shown) arranged in the cavity 11. The droplet 210 can be ejected from the nozzle 12 in the ejection direction shown by D3 in FIG.

The driving means 20 is electrically connected to the piezoelectric element of the droplet discharging means 10, and a driving voltage is applied to the piezoelectric element to deform the piezoelectric element to discharge the droplet 210.

The light irradiation means 30 is electrically connected to the drive means 20, and a synchronization signal is input from the drive means 20. The light irradiating means 30 to which the synchronization signal is input irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210 by the droplet discharging means 10.

Both the two light receiving means 41 and 42 are electrically connected to the driving means 20 via the light irradiating means 30, and a synchronization signal is input from the driving means 20. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiation means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface, respectively.

The particle counting means 51 is a liquid based on information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 received by the two light receiving means 41 and 42, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. The fluorescent particles 201 contained in the droplet 210 are counted.

FIG. 2 is an explanatory diagram showing an example of the positional relationship between the two light receiving means in the particle counting device shown in FIG. In FIG. 2, the ejection direction of the droplet is the depth direction of the paper surface, and the adherend 300 is arranged directly below the nozzle 12.
As shown in FIG. 2, the light irradiation means 30, the mirror 60, and the lens 70 are liquids in which the laser beam L irradiated by the light irradiation means 30 is reflected by the mirror 60 and discharged in the discharge direction through the lens 70. It is arranged so as to be focused on droplets (position of the nozzle 12 in FIG. 2).
The two light receiving means 41 and 42 are arranged on a plane whose normal is the discharge direction, with an angle formed by the light receiving direction D1 of the light receiving means 41 and the light receiving direction D2 of the light receiving means 42 being 90 °.

FIG. 3 is an explanatory diagram of an image of light emission obtained by the two light receiving means having the positional relationship shown in FIG. In FIG. 3, the brightness of the light emitted by the light receiving means 41 and the shape of the light emitting surface on the light receiving surface are shown in one image P1, and the brightness of the light emitted by the light receiving means 42 and the shape of the light emitting surface on the light receiving surface are shown in one image P2. Shown.
As shown in FIG. 3, the light emission does not overlap in the image P1, but the light emission overlaps in the image P2.
When counting the fluorescent particles 201 from the image P1 and the image P2 based on the brightness of light emission, either the above processing (1) or (2) may be performed to count the number of fluorescent particles 201.
When counting the particles 201 from the image P1 and the image P2 based on the shape of the light emitting surface on the light receiving surface, any of the above processes (3) to (6) may be performed to count the number of fluorescent particles 201.

4A to 4C are explanatory views showing an example of a simulation image of the luminance value of the emission and the shape of the light receiving surface of the emission when the two emissions overlap. In FIGS. 4A to 4C, particles that can transmit light emission are assumed, and the shade of the image indicates the brightness of the light emission, and the lighter the color, the higher the brightness of the light emission.

As shown in FIG. 4A, the number of particles can be determined based on the brightness value of the light emission as long as the light emission from the particles overlaps.
As shown in FIG. 4B, in the case of overlapping light emission from particles, when the number of particles cannot be determined based on the brightness value of light emission, the curvature of the shape on the light receiving surface of light emission is based on the shape of the light receiving surface of light emission. The radius is calculated, and the number of centers of the calculated radius of curvature is determined as the number of particles.
As shown in FIG. 4C, even when the light emission from the particles completely overlaps, it can be determined that the number of overlapping particles increases as the brightness increases, so that the number of particles can be determined using the calibration curve. ..
Even if the particles do not transmit light, as long as the particles can receive light in a state where the light emission does not overlap by having two or more light receiving means, unless the particles are in contact with each other and overlap. The number of particles can be determined based on the brightness value of light emission.

5A and 5B are explanatory views showing an example of a simulation image of the brightness of light emission and the shape on the light receiving surface of light emission when the particles are present in the vicinity of the outer edge portion of the droplet. In FIGS. 5A and 5B, similarly to FIGS. 4A to 4C, the shade of the image indicates the brightness of the light emission, and the lighter the color, the higher the brightness of the light emission. However, it differs from FIGS. 4A to 4C in that one particle is contained in the droplet and the particle exists near the outer edge of the droplet.
As shown in FIG. 5A, the outer edge of the droplet emits light, and when trying to determine the number of particles based on the brightness value of the emission, it is determined whether the number of particles is one or two. Although it is difficult, if the number of particles is determined based on the shape of the light emitting surface on the light receiving surface, it can be excluded because the radius of curvature of the outer edge of the droplet is large.
As shown in FIG. 5B, when the number of particles is determined based on the luminance value of the light emission, it is difficult to determine whether the number of particles is one or two, as in the case of FIG. 5A. However, when the number of particles is determined based on the shape of the light emitting surface on the light receiving surface, it can be determined that the number of particles is one from the radius of curvature of the emitted light.

6A to 6C are images showing an example of the brightness value of the light emitted by the light receiving means and the shape of the light emitting surface on the light receiving surface. 6A to 6C show SPHERO Fluorescent Nile Red spheres (manufactured by Bay Biosciences Co., Ltd., 1% (w / v), diameter 10 μm to 14 μm) as particles, and ion-exchanged water as a liquid component of the suspension. A membrane vibration type inkjet head using a piezoelectric element is driven by a piezoelectric element with a sinusoidal voltage (20,000 Hz, 1 cycle, 4.6 V) to eject droplets containing particles into a sphere having a diameter of 80 μm. , The exposure time of a high-sensitivity camera (pco.edge, manufactured by Tokyo Instruments Co., Ltd.) was set to 10 ms as a light receiving means, and the YAG laser (Explorer ONE-532-200-KE) as a light irradiating means for ejected droplets It is an image of a state in which particles are emitted by irradiating a laser beam (100 μJ at 10 kHz) emitted by Spectra Physics Co., Ltd.). The inkjet head, the YAG laser, and the high-sensitivity camera were synchronized, and the delay time was set to 0.1 ms so as to delay only the YAG laser. However, the above conditions are not particularly limited as long as an image can be obtained, and can be appropriately selected depending on the equipment used, the physical characteristics of the discharged liquid, the fluorescent particles, and the like.

In FIG. 6A, two particles are present in a deep spherical or ellipsoidal droplet, one of the particles is on the front side of the paper surface in the droplet, and the other particle is in the droplet. It is an image when the light is received from the light receiving direction as if it exists on the back side of the paper surface.
As shown in FIG. 6A, the light emission from the particles existing on the back side of the paper surface in the droplet has the luminance value of the light emission attenuated by refraction or scattering as much as it passes through the droplet, so that the liquid for the light receiving means. The brightness value of light emission may vary depending on the position in the droplet.

FIG. 6B is an image when three particles are present in a deep spherical or ellipsoidal droplet and one of the particles is received from a light receiving direction such that one of the particles is near the outer edge of the droplet. is there.
As shown in FIG. 6B, light emitted from particles existing near the outer edge of the droplet may be reflected inside the spherical surface of the droplet.

FIG. 6C shows a case where one particle is present in a deep spherical or ellipsoidal droplet and the particle is received from a light receiving direction such that the particle is near the outer edge of the droplet on the inner side of the paper surface. It is an image.
As shown in FIG. 6C, when the particles are present near the outer edge of the droplet on the inner side of the paper surface, the brightness value of the light emitted from the particles is low, and the shape on the light receiving surface may be unclear.

FIG. 7 is a graph showing an example of a calibration curve for determining the number of particles based on the luminance value of the light emitted by two or more light receiving means.
As shown in FIG. 7, by using a calibration curve that determines the number of particles based on the luminance value of the emission, the luminance value 1 of the emission received by the first light receiving means and the emission received by the imaging means are used. The number of particles can be determined based on the brightness value 2.
Further, as shown in FIGS. 6A to 6C, the luminance value of the light emission becomes unclear and the calibration curve may not be obtained. In this case, the number of particles is determined in a predetermined region near the boundary line. An area that cannot be determined is provided as an area that cannot be determined. When plotting in a non-determinable region based on the brightness value received by the two light receiving means, it is determined that the particles cannot be counted only by the brightness value, and the particles are counted based on the shape information on the light receiving surface of the light emission. It is preferable to do so.
The calibration curve data is acquired in advance according to the type of particles, light emission conditions, etc., and stored in a storage means or the like.

Next, using the particle counting device of the present invention, the flow of counting the particles contained in the droplets ejected from the droplet ejection means is shown in FIG. 1 according to the step represented by S in the flowchart of the flowchart shown in FIG. Will be explained with reference to.
This flowchart is a procedure used when the light emission does not overlap, or when the light emission is not reflected inside the spherical surface of the droplet and becomes unclear.

In S101, the droplet ejection means 10 to which the driving voltage is applied from the driving means 20 ejects the droplet 210 containing the fluorescent particles 201 from the nozzle 12 in the ejection direction shown by D3 in FIG. Further, the driving means 20 applies a driving voltage to the droplet discharging means 10 and outputs a synchronization signal to the light irradiating means 30 and the two light receiving means 41 and 42. After that, the particle counting means 51 shifts the processing to S102.

In S102, the droplet ejection means 10 to which the synchronization signal is input from the driving means 20 irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210. Further, the light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiating means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. After that, the particle counting means 51 shifts the processing to S103.

In S103, the particle counting means 51 receives information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, the shape of the light emitting L1 on the light receiving surface, and the shape of the light emitting L2 on the light receiving surface, which are received by the two light receiving means 41 and 42. Based on the above, the fluorescent particles 201 contained in the droplet 210 are counted using the calibration curve shown in FIG. 7, and this process is completed.

Next, using the particle counting device of the present invention, the flow of counting the particles contained in the droplets ejected from the droplet ejection means is shown in FIG. 1 according to the step represented by S in the flowchart of the flowchart shown in FIG. Will be explained with reference to.
This flowchart is a procedure used, for example, when the number of fluorescent particles 201 contained in the droplet 210 ejected by the droplet ejection means 10 varies from ejection to ejection. That is, in order to confirm the degree of variation in the number of the fluorescent particles 201, the brightness value of the light emitting L1 and the brightness value of the light emitting L2 from the fluorescent particles 201, the shape of the light receiving surface of the light emitting L1 and the light receiving surface of the light emitting L2 each time the fluorescence particles 201 are discharged. This is a procedure for counting the fluorescent particles 201 based only on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 by obtaining the shape information in the above.

In S201, the droplet ejection means 10 to which the driving voltage is applied from the driving means 20 ejects the droplet 210 containing the fluorescent particles 201 from the nozzle 12 in the ejection direction shown by D3 in FIG. Further, the driving means 20 applies a driving voltage to the droplet discharging means 10 and outputs a synchronization signal to the light irradiating means 30 and the two light receiving means 41 and 42. After that, the particle counting means 51 shifts the processing to S202.

In S202, the droplet ejection means 10 to which the synchronization signal is input from the driving means 20 irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210. Further, the light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiating means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. After that, the particle counting means 51 shifts the processing to S203.

In S203, the particle counting means 51 uses the calibration curve shown in FIG. 7 to fluoresce the droplet 210 based on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 received by the two light receiving means 41 and 42. Particle 201 is counted. After that, the particle counting means 51 shifts the processing to S204.

In S204, the particle counting means 51 that counts the fluorescent particles 201 contained in the droplet 210 determines whether or not the droplet discharging means 10 has discharged the droplet 210 a predetermined number of times. When the particle counting means 51 determines that the droplet discharging means 10 has discharged the droplet 210 a predetermined number of times, the particle counting means 51 ends this process. When the particle counting means 51 determines that the droplet discharging means 10 has not discharged the droplet 210 a predetermined number of times, the process returns to S201.

Next, using the particle counting device of the present invention, the flow of counting the particles contained in the droplets ejected from the droplet ejection means is shown in FIG. 1 according to the step represented by S in the flowchart of the flowchart shown in FIG. Will be explained with reference to.
This flowchart is a procedure used, for example, when it is desired to confirm how much fluorescent particles 201 are contained in the continuously ejected droplet 210 after the droplet 210 is continuously ejected by the droplet ejection means 10. Is.

In S301, the droplet ejection means 10 to which the driving voltage is applied from the driving means 20 ejects the droplet 210 containing the fluorescent particles 201 from the nozzle 12 in the ejection direction shown by D3 in FIG. Further, the driving means 20 applies a driving voltage to the droplet discharging means 10 and outputs a synchronization signal to the light irradiating means 30 and the two light receiving means 41 and 42. After that, the particle counting means 51 shifts the processing to S302.

In S302, the droplet ejection means 10 to which the synchronization signal is input from the driving means 20 irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210. Further, the light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiating means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. The particle counting means 51 stores information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, the shape of the light emitting L1 on the light receiving surface, and the shape of the light emitting L2 on the light receiving surface in a storage means (not shown) for each droplet 210. The process shifts to S303.

In S303, the particle counting means 51 that counts the fluorescent particles 201 contained in the droplet 210 determines whether or not the droplet discharging means 10 has discharged the droplet 210 a predetermined number of times. When the particle counting means 51 determines that the droplet discharging means 10 has discharged the droplet 210 a predetermined number of times, the process shifts to S304. When the particle counting means 51 determines that the droplet discharging means 10 has not discharged the droplet 210 a predetermined number of times, the process returns to S301.

In S304, the particle counting means 51 that determines that the droplet 210 has been ejected a predetermined number of times has the brightness value of the light emitting L1 and the brightness value of the light emitting L2 stored in the storage means, the shape of the light emitting L1 on the light receiving surface, and the light emitting L2. Based on the shape information on the light receiving surface, the fluorescent particles 201 are counted for each droplet 210 using the calibration curve shown in FIG. 7, and this process is completed.

Next, using the particle counting device of the present invention, the flow of counting the particles contained in the droplets ejected from the droplet ejection means is shown in FIG. 1 according to the step represented by S in the flowchart of the flowchart shown in FIG. Will be explained with reference to.
When it is determined that the number of fluorescent particles 201 cannot be counted based only on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, this flowchart is based on the shape of the light receiving surface of the light emitting L1 and the shape of the light receiving surface of the light emitting L2. This is a procedure for determining the number of fluorescent particles 201.

In S401, the droplet ejection means 10 to which the driving voltage is applied from the driving means 20 ejects the droplet 210 containing the fluorescent particles 201 from the nozzle 12 in the ejection direction shown by D3 in FIG. Further, the driving means 20 applies a driving voltage to the droplet discharging means 10 and outputs a synchronization signal to the light irradiating means 30 and the two light receiving means 41 and 42. After that, the particle counting means 51 shifts the processing to S402.

In S402, the droplet ejection means 10 to which the synchronization signal is input from the driving means 20 irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210. Further, the light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiating means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. After that, the particle counting means 51 shifts the processing to S403.

In S403, the particle counting means 51 uses the calibration curve shown in FIG. 7 to obtain fluorescent particles for each droplet 210 based on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 received by the two light receiving means 41 and 42. The number of 201 is determined. At this time, when the particle counting means 51 determines that the brightness value of the received light emitting L2 is not included in the undeterminable region shown in FIG. 7, the process shifts to S404. When the particle counting means 51 determines that it is included in the undeterminable region, the process shifts to S405.

In S404, the particle counting means 51 determined not to be included in the undeterminable region is the calibration curve shown in FIG. 7 based on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 received by the two light receiving means 41 and 42. The number of fluorescent particles 201 contained in the droplet 210 is counted using the above, and this process is completed.

In S405, the particle counting means 51 determined to be included in the undeterminable region provides information on the shape of the light emitting L1 received by the two light receiving means 41 and 42 on the light receiving surface and the shape information of the light emitting L2 on the light receiving surface, that is, shape data. Based on this, the radius of curvature is obtained.
Next, the particle counting means 51 determines whether or not the obtained radius of curvature is within a predetermined range. When the particle counting means 51 determines that all the obtained radius of curvature is within a predetermined range, the process shifts to S406, and when it is determined that even one of the obtained radius of curvature is not within the predetermined range, the process is performed. Move to S404.

In S406, the particle counting means 51, which determines that all the radius of curvature is within a predetermined range, counts the number of centers of the determined radius of curvature as the number of fluorescent particles 201 contained in the droplet 210, and performs this process. finish.

Next, using the particle counting device of the present invention, the flow of counting the particles contained in the droplets ejected from the droplet ejection means is shown in FIG. 1 according to the step represented by S in the flowchart of the flowchart shown in FIG. It will be explained with reference to it.

In S501, the droplet ejection means 10 to which the driving voltage is applied from the driving means 20 ejects the droplet 210 containing the fluorescent particles 201 from the nozzle 12 in the ejection direction shown by D3 in FIG. Further, the driving means 20 applies a driving voltage to the droplet discharging means 10 and outputs a synchronization signal to the light irradiating means 30 and the two light receiving means 41 and 42. After that, the particle counting means 51 shifts the processing to S502.

In S502, the droplet ejection means 10 to which the synchronization signal is input from the driving means 20 irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210. Further, the light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiating means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. After that, the particle counting means 51 shifts the processing to S503.

In S503, the particle counting means 51 uses the calibration curve shown in FIG. 7 to fluoresce the droplet 210 based on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 received by the two light receiving means 41 and 42. Particle 201 is counted. After that, the particle counting means 51 shifts the processing to S504.

In S504, the particle counting means 51 determines whether or not the counted fluorescent particles 201 are 0. When the particle counting means 51 determines that the number of counted fluorescent particles 201 is 0, the process shifts to S501. When the particle counting means 51 determines that the number of counted fluorescent particles 201 is not 0, the particle counting means 51 ends this process.

Next, when the particle counting means determines that the number of particles contained in the discharged droplets is 0 by using the particle counting device of the present invention, the droplet discharging means re-discharges the droplets and fluoresces. The flow of repeating until the particles 201 are counted will be described with reference to FIG. 1 according to the step represented by S in the flowchart of the flowchart shown in FIG.

In S601, the droplet ejection means 10 to which the driving voltage is applied from the driving means 20 ejects the droplet 210 containing the fluorescent particles 201 from the nozzle 12 in the ejection direction shown by D3 in FIG. Further, the driving means 20 applies a driving voltage to the droplet discharging means 10 and outputs a synchronization signal to the light irradiating means 30 and the two light receiving means 41 and 42. After that, the particle counting means 51 shifts the processing to S602.

In S602, the droplet ejection means 10 to which the synchronization signal is input from the driving means 20 irradiates the droplet 210 with laser light L as light in accordance with the ejection timing of the droplet 210. Further, the light receiving means 41 and 42 to which the synchronization signal is input receive the light emitting L1 and the light emitting L2 at the timing when the fluorescent particles 201 emit light by the laser light L of the light irradiating means 30. As a result, the light receiving means 41 and 42 obtain information on the brightness value of the light emitting L1 and the brightness value of the light emitting L2, and the shape of the light emitting L1 on the light receiving surface and the shape of the light emitting L2 on the light receiving surface. After that, the particle counting means 51 shifts the processing to S603.

In S603, the particle counting means 51 uses the calibration curve shown in FIG. 7 to fluoresce the droplet 210 based on the brightness value of the light emitting L1 and the brightness value of the light emitting L2 received by the two light receiving means 41 and 42. Particle 201 is counted. After that, the particle counting means 51 shifts the processing to S604.

In S604, the particle counting means 51 that counts the fluorescent particles 201 determines whether or not the counted light particles 201 are 0. When the particle counting means 51 determines that the number of counted light particles 201 is 0, the process shifts to S605. When the particle counting means 51 determines that the number of counted light particles 201 is not 0, the particle counting means 51 ends this process.

In S605, the particle counting means 51 discharges the droplet 210 determined to have 0 fluorescent particles 201 so as not to adhere to the adherend, and returns the process to S601.

14 and 15 are explanatory views showing an embodiment different from the embodiment of the particle counting device of the present invention described above.

FIG. 14 is an explanatory diagram showing another example of the particle counting device of the present invention. As shown in FIG. 14, similarly to the particle counting device shown in FIG. 1, the droplet 210 is ejected and landed on the adhered object 71, but the fluorescent particles 201 landing on the adhered object 71 are landed on the adhered object 71. The difference is that counting is performed by the second particle counting means 52 from the bottom and the result is fed back to the droplet discharging means 10.
The image pickup means 80 is provided with an objective lens 83 at a position where the landed droplets can be observed. The laser beam L emitted from the second light irradiating means 81 of the imaging means 80 passes through the objective lens 83 via the dichroic mirror 83 and irradiates the fluorescent particles in the well as an adherend. Image information can be acquired by the fluorescence L3 emitted from the fluorescent particles passing through the objective lens 63 and incident on the image sensor 65.

FIG. 15 is an explanatory diagram showing still another example of the particle counting device of the present invention. As shown in FIG. 15, similarly to the particle counting device shown in FIG. 1, the droplets are ejected and landed on the adherend 71, but the droplet 210 containing the fluorescent particles 201 at a predetermined position of the adherend 71. After landing, the adherend 71 is moved on the stage 72 to the place where the objective lens 83 of the imaging means 80 is installed so that the landed droplets can be observed, and the fluorescence landed on the adhered object 71. The difference is that the particles 201 are counted from above the adherend 71 by the particle number counting means 52, and the result is fed back to the droplet ejection means 10.

FIG. 16 is a flowchart showing an example of a flow in which the number of particles contained in the droplets landing on the adherend is set to a predetermined number by using the particle counting device of the present invention. As shown in FIG. 16, the flow is the same as that in FIG. 12, but the “determination of whether or not the number of particles is 0” in step S504 of FIG. 12 is determined by “the number of particles is a predetermined number”. A flowchart is shown in the case where a predetermined number of particles are landed on the adherend instead of "determination of whether or not".

FIG. 17 is a flowchart showing another example of a flow in which the number of particles contained in the droplets landing on the adherend is set to a predetermined number by using the particle counting device of the present invention. Similar to FIG. 16, a flow chart in the case of landing a predetermined number on the adherend is shown, and a flowchart in the case of discharging droplets when the number of particles becomes a predetermined number or more is shown.

Examples of aspects of the present invention are as follows.
<1> A droplet ejection means for ejecting droplets containing particles capable of emitting light when irradiated with light, and a light irradiation means for irradiating the droplets ejected from the droplet ejection means with the light. Two or more light receiving means for receiving light emitted from the particles irradiated with the light, and a particle counting means for counting the particles contained in the droplet based on the light emitted by the two or more light receiving means. It is a particle counting device characterized by having.
<2> The particle counting means obtains the brightness value of the light emission received by the light receiving means in the substantially normal direction of the light receiving surface and the shape information of the light emitting surface on the light receiving surface, and the particle counting means determines the brightness value of the light emission. A first counting process for counting the particles contained in the droplets based on the above, and a second counting process for counting the particles contained in the droplets based on the shape information on the light receiving surface of the light emission. The particle counting device according to <1>, which performs at least one of the above.
<3> The particles according to <2>, wherein when the particle counting means performs the first counting process and determines that the particles cannot be counted, the second counting process is performed and the particles are counted. Counting device.
<4> The number of particles is the number of centers of the circle when the particle counting means calculates the radius of curvature based on the shape information on the light receiving surface of the light emission and calculates the radius of curvature within a predetermined range. The particle counting device according to any one of <2> to <3>, which performs the second counting process.
<5> The light receiving means of at least one of the two or more light receiving means is arranged so that the light receiving direction thereof is located substantially orthogonal to the light receiving direction of the other light receiving means. > Is the particle counting device according to any one of.
<6> The particle counting device according to any one of <1> to <5>, wherein the light receiving means is arranged so that the light receiving direction is located in a direction substantially orthogonal to the ejection direction of the droplet.
<7> The particle counting device according to any one of <1> to <6>, wherein the light receiving means is a camera having a CMOS image sensor.
<8> When the particle counting means determines that the number of the particles is 0, the droplet discharging means further discharges the droplets according to any one of <1> to <7>. It is a particle counting device of.
<9> When the particle counting means determines that the number of particles is less than a predetermined number, the droplet discharging means further discharges the droplets from any of <1> to <7>. The particle counting device according to the above.
<10> The particle counting device according to any one of <1> to <7>, wherein when the particle counting means determines that the number of particles is a predetermined number, the process proceeds to the next step.
<11> The particle counting device according to any one of <1> to <10>, wherein the droplet discharging means is an inkjet head by a piezoelectric pressurizing method using a piezoelectric element.
<12> The particle counting device according to <11>, wherein the piezoelectric element is an element using lead zirconate titanate.
<13> The particle counting device according to any one of <1> to <12>, wherein the light irradiation means is a solid-state laser.
<14> The particle counting device according to any one of <1> to <13>, wherein the light is pulsed light.
<15> The particle counting device according to <14>, wherein the pulse width of the pulsed light is 10 μs or less.
<16> The particle counting device according to any one of <14> to <15>, wherein the energy per unit pulse in the pulsed light is 0.1 μJ or more.
<17> A droplet ejection step of ejecting droplets containing particles capable of emitting light when irradiated with light, a light irradiation step of irradiating the ejected droplets with the light, and irradiation with the light. It includes a light receiving step of receiving light emitted from the particles by two or more light receiving means and a particle counting step of counting the particles contained in the droplet based on the light received by two or more light receiving means. It is a particle counting method characterized by.

The particle counting device according to any one of <1> to <16> and the particle counting method according to <17> can solve the above-mentioned problems in the past and achieve the object of the present invention. ..

10 Droplet ejection means 20 Driving means 30, 81 Light irradiation means 41, 42 Light receiving means 51 Particle counting means 52 Second particle counting means 71 Adhesion 80 Imaging means 100 Particle counting device 201 Fluorescent particles 210 Droplets

Japanese Patent No. 5716213

Claims (10)

A droplet ejection means that ejects droplets containing particles that can emit light when irradiated with light,
A light irradiation means for irradiating the droplets ejected from the droplet ejection means with the light,
Two or more light receiving means for receiving light emission from the particles irradiated with the light, and
A particle counting means for counting the particles contained in the droplet based on the light emission received by the two or more light receiving means, and a particle counting means.
Have a,
The light receiving means obtains information on the brightness value of the light emitted received in the substantially normal direction of the light receiving surface and the shape of the light emitted on the light receiving surface.
When the particle counting means performs the first counting process of counting the particles contained in the droplets based on the brightness value of the light emission and determines that the particles cannot be counted, the light receiving surface of the light emission. A particle counting device characterized in that a second counting process for counting the particles contained in the droplets is performed based on the shape information, and the particles are counted. The particle counting means calculates the radius of curvature based on the shape information on the light receiving surface of the light emission, and counts the number of centers of the circles within a predetermined range as the number of the particles. The particle counting device according to claim 1 , wherein the second counting process is performed. At least one of said light receiving means of the two or more of said light receiving means, according to claim 1 which is adapted to be positioned in the receiving direction substantially perpendicular direction of 2 of the light-receiving direction other of said light receiving means Particle counting device. The particle counting device according to any one of claims 1 to 3 , wherein the light receiving means is arranged so that the light receiving direction is located in a direction substantially orthogonal to the ejection direction of the droplet. The particle counting device according to any one of claims 1 to 4 , wherein the light receiving means is a camera having a CMOS image sensor. When the particle counting means determines that the number of particles is 0,
The particle counting device according to any one of claims 1 to 5 , wherein the droplet discharging means further discharges the droplet. When the particle counting means determines that the number of particles is less than a predetermined number,
The particle counting device according to any one of claims 1 to 5 , wherein the droplet discharging means further discharges the droplet. The particle counting device according to any one of claims 1 to 7 , wherein when the particle counting means determines that the number of particles is a predetermined number, the process proceeds to the next step. A droplet ejection process that ejects droplets containing particles that can emit light when irradiated with light,
A light irradiation step of irradiating the discharged droplets with the light,
A light receiving step of receiving light emitted from the particles irradiated with the light by two or more light receiving means, and a light receiving step.
A particle counting step of counting the particles contained in the droplet based on the light emitted by the two or more light receiving means.
Only including,
In the light receiving step, information on the brightness value of the light emitted received in the substantially normal direction of the light receiving surface and the shape of the light emitted on the light receiving surface is obtained.
In the particle counting step, when the first counting process of counting the particles contained in the droplets is performed based on the brightness value of the light emission and it is determined that the particles cannot be counted, the light receiving surface of the light emission. A particle counting method characterized in that a second counting process for counting the particles contained in the droplets is performed based on the shape information, and the particles are counted. A droplet ejection process that ejects droplets containing particles that can emit light when irradiated with light,
A light irradiation step of irradiating the discharged droplets with the light,
A light receiving step of receiving light emitted from the particles irradiated with the light by two or more light receiving means, and a light receiving step.
A particle counting step of counting the particles contained in the droplet based on the light emitted by the two or more light receiving means.
A step of adhering the discharged droplets to an adherend on which a well is formed to form a well containing a preset number of particles with respect to the well.
Including
In the light receiving step, information on the brightness value of the light emitted received in the substantially normal direction of the light receiving surface and the shape of the light emitted on the light receiving surface is obtained.
In the particle counting step, when the first counting process of counting the particles contained in the droplets is performed based on the brightness value of the light emission and it is determined that the particles cannot be counted, the light receiving surface of the light emission. A method characterized in that a second counting process for counting the particles contained in the droplets is performed based on the shape information, and the particles are counted.

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