JP6915240B2 - Particle counting device and particle counting method, as well as droplet forming device and dispensing device - Google Patents

Description

The present invention relates to a particle counting device, a particle counting method, a droplet forming device, and a dispensing device.

In recent years, with the progress of stem cell technology, a technique for ejecting a plurality of cells by inkjet to form a tissue has been developed. When ejecting a droplet containing particles typified by cells, it is important to detect how many particles are contained in the ejected droplet.

As a device having such a function, for example, a discharge head that pressurizes the diaphragm of the liquid chamber with a piezoelectric element and discharges a liquid containing cells from a nozzle is provided, and the discharged droplets are irradiated with laser light. However, a discharge device for measuring the number of cells in a droplet discharged by photographing with a CCD camera or a photodiode or measuring the amount of light has been proposed (see, for example, Patent Document 1).

The present invention has high productivity in which the detection accuracy of particles contained in the ejected droplets is improved and the number of particles in the droplets can be detected even if the number of droplets ejected per unit time is increased. It is an object of the present invention to provide a particle counting device.

The particle counting device of the present invention as a means for solving the above-mentioned problems contains particles capable of emitting light when irradiated with light, and irradiates a plurality of droplets ejected from different positions with light. Light irradiation means and
A light receiving means that receives light emitted from particles irradiated with light,
It has a particle counting means for counting particles contained in a plurality of droplets based on the light emitted by the light receiving means.

According to the present invention, the detection accuracy of the particles contained in the ejected droplets is improved, and the number of particles in the droplets can be detected even if the number of droplets ejected per unit time is increased. A particle counting device having the same can be provided.

FIG. 1 is a schematic view showing an example of the droplet forming apparatus of the present invention. FIG. 2 is a schematic view showing another example of the droplet forming apparatus of the present invention. FIG. 3 is a schematic view showing another example of the droplet forming apparatus of the present invention. FIG. 4 is a schematic view showing another example of the droplet forming apparatus of the present invention. FIG. 5 is a schematic view showing another example of the droplet forming apparatus of the present invention. FIG. 6 is a schematic view showing another example of the droplet forming apparatus of the present invention. FIG. 7 is a schematic plan view showing an example of an arrangement state of a plurality of ejection ports on the ejection surface of the droplet ejection means in the droplet forming apparatus of the present invention. FIG. 8 is a schematic plan view showing another example of the arrangement state of the plurality of ejection ports on the ejection surface of the droplet ejection means in the droplet forming apparatus of the present invention. FIG. 9 is a schematic plan view showing another example of the arrangement state of the plurality of ejection ports on the ejection surface of the droplet ejection means in the droplet forming apparatus of the present invention. FIG. 10 is a schematic plan view showing another example of the arrangement state of the plurality of ejection ports on the ejection surface of the droplet ejection means in the droplet forming apparatus of the present invention. FIG. 11 is a schematic plan view showing another example of the arrangement state of the plurality of ejection ports on the ejection surface of the droplet ejection means in the droplet forming apparatus of the present invention. FIG. 12 is an explanatory diagram showing an example of the positional relationship between the two light receiving means in the droplet forming apparatus shown in FIG. FIG. 13 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. 14A 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. 14B 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. 14C 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. 15A is an explanatory diagram showing an 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. 15B 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. 16A 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. 16B 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. 16C 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. 17 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. 18 is a flowchart showing an example of a flow for counting the particles contained in the droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention. FIG. 19 is a flowchart showing another example of a flow of counting the particles contained in the droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention. FIG. 20 is a flowchart showing another example of a flow of counting the particles contained in the droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention. FIG. 21 is a flowchart showing another example of a flow of counting the particles contained in the droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention. FIG. 22 is a flowchart showing another example of a flow of counting the particles contained in the droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention. FIG. 23 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 droplet forming apparatus of the present invention. FIG. 24 is a schematic view showing an example of the dispensing device of the present invention. FIG. 25 is a flowchart showing an example of the operation of the dispensing device of the present invention. FIG. 26 is a flowchart showing another example of the operation of the dispensing device of the present invention.

(Particle counting device and particle counting method)
The particle counting device of the present invention includes a light irradiating means, a light receiving means, a particle counting means, and further has other means as needed.

The particle counting method of the present invention includes a light irradiation step, a light receiving step, a particle counting step, and further includes other steps as necessary.

The particle counting method of the present invention can be suitably carried out by the particle counting device of the present invention, the light irradiation step can be performed by the light irradiation means, the light receiving step can be performed by the light receiving means, and the particle counting step. Can be performed by particle counting means, and other steps can be performed by other means.
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.

The particle counting device of the present invention has a conventional single nozzle (one ejection port), and the ejection device described in Patent Document 1 has a small number of droplets to be ejected per unit time, resulting in low productivity and particles. As a result, droplets containing cells having a short lifetime are ejected, which is disadvantageous in forming a cell tissue within a limited time in which the cells are alive. Since cells that do not emit light when irradiated with light are irradiated with laser light to measure the optical image and the amount of light, and the cells in the droplets are counted, the detection accuracy is low and false detection may occur. It is based on the knowledge.

<Light irradiation means and light irradiation process>
The light irradiation means is a means for irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejecting light from different positions.
The light irradiation step is a step of irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejected from different positions, and may be preferably performed by a light irradiation means. can.

The different positions mean that the ejection start points of the plurality of droplets to be ejected are different, that is, there are a plurality of ejection start points of the droplets. When the particle counting device is a droplet forming device having a droplet ejection means, the plurality of ejection start points correspond to a plurality of ejection ports described later.
As the plurality of ejection start points, it is preferable that one or more rows are arranged along the direction orthogonal to the ejection direction of the plurality of droplets, and one row or more and four rows or less are more preferable.
The number of discharge start points per row is not particularly limited and is appropriately selected according to the purpose, but 2 or more and 100 or less are preferable, 2 or more and 50 or less are more preferable, and 2 or more and 12 are preferable. More preferably, the number is less than one.

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.).
The spot diameter of the laser is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 100 μm or more and 2,000 μm or less. If the spot diameter is 100 μm or more and 2,000 μm or less, the probability that the droplets will be irradiated with the laser will increase even if the ejection variation of the flying droplets occurs, so that the counting accuracy of the particles in a plurality of droplets will decrease. Has the advantage of being able to suppress.

The light emitted from the light irradiating means is preferably pulsed light. As a result, the accuracy of counting the number of particles in the droplet can be improved.
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. ..

The light irradiation means irradiates a plurality of flying droplets with light. In addition, “in flight” refers to a state from the time when the droplet is ejected until the droplet is deposited on the object to be adhered.
As the light irradiation means, it is preferable that light can be irradiated in synchronization with the ejection of a plurality of droplets. As a result, it is possible to more reliably irradiate a plurality of droplets ejected from different positions with light.
Here, "synchronizing" means that the light irradiating means irradiates 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.
The light emitted from the light irradiation means preferably irradiates one flying droplet. When multiple droplets are ejected from different positions at the same time and the light is irradiated to the plurality of droplets at the same time, the light emitted from the particles that can emit light when the light contained in the multiple droplets is irradiated is simultaneously received. Therefore, it becomes difficult to count the number of particles contained in each droplet. That is, in order to improve the accuracy of counting the number of particles in the droplet, it is preferable to shift the timing at which a plurality of droplets are ejected from different positions.
Further, even if a plurality of flying droplets are simultaneously irradiated with light, for example, by using a light receiving means in which a lens and a plurality of light receiving elements are arranged in an array, the particles of the plurality of droplets are counted at the same time. can do.

The particles capable of emitting light when irradiated with light are not particularly limited and may be appropriately selected depending on the intended purpose, but cells are preferable, and the number of particles in the flying droplets is counted by receiving autofluorescence. From the possible points, cells expressing fluorescent protein, stained cells stained with fluorescent dye, inorganic fine particles stained with fluorescent dye, and organic polymer particles stained with fluorescent dye are preferable, and cells expressing fluorescent protein and fluorescent dye. Stained cells stained with are particularly preferred.

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.
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 number of particles contained in the droplet is preferably 1 or more, and more preferably 1 or more and 5 or less. If the droplet does not contain cells as particles, the tissue in that part will be lost. If the droplets contain an excessive amount of cells as particles, oxygen and nutrients may be deficient and the cell retention rate may decrease.

When the particles agglomerate, the concentration of the particles in the liquid and the number of particles in the liquid follow the Poisson distribution by adjusting the concentration of the particles in the liquid including the particles. The number of particles can be adjusted as appropriate.
The liquid is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ion-exchanged water, distilled water, pure water, and physiological saline.

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 contained particles becomes appropriate, and the types of particles that can be applied increase. Further, when the diameter of the droplet is 150 μm or less, the ejection of the droplet becomes stable.
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, the relationship between the particle diameter and the droplet diameter is appropriate and is not affected by the droplet edge, so that the particle counting accuracy is improved.
The amount of the liquid droplet is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1,000 pL or less, more preferably 100 pL or less.
The liquid amount of the droplet can be measured, for example, by obtaining the size of the droplet from the image of the droplet and calculating the liquid amount.

<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, and can be preferably performed by the light receiving means.

The light receiving means is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a one-dimensional element and a camera having a two-dimensional element. 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.
Examples of commercially available cameras having a CMOS image sensor include high-sensitivity cameras (pco.edge, sCMOS, manufactured by Tokyo Instruments Co., Ltd.).

The light receiving means receives fluorescence emitted by absorbing light as excitation light when particles capable of emitting light are contained when a plurality of flying droplets are irradiated with light. Since the fluorescence is emitted from the particles in all directions, the light receiving means can be arranged at an arbitrary position where the light emitted from the particles can be received. At this time, 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.

It is preferable that two or more light receiving means are provided, and each light receiving means receives light emitted from particles from different directions. By having two or more light receiving means, even if one light receiving means receives the state in which the light emission overlaps, if the other light receiving means can receive the state in which the light emission does not overlap, the other light receiving means can receive the light. The particles contained in the droplet can be accurately counted based on the light emitted by the light receiving means.

Since light emission is emitted from particles capable of emitting light in all directions when irradiated with light, two or more light receiving means receive light emitted from particles capable of emitting light in different directions when irradiated with light. It can be placed in any position where it can be placed. It should be noted that three or more light receiving means may be arranged at positions where light emitted from particles capable of emitting light when irradiated with light can be received in different directions. Further, each light receiving means may have the same specifications or may have different specifications.
When there is only one light receiving means, when the flying droplets contain particles that can emit light when a plurality of lights are irradiated, the particles that can emit light when irradiated with light overlap each other. As a result, the particle counting means may erroneously detect the number of particles capable of emitting light when the light contained in the droplet is irradiated (a counting error occurs), but two or more light receiving means are provided. This makes it possible to reduce the effect of overlapping particles that can emit light when irradiated with light.
The particle counting means described later can be executed by comparing the brightness value or area value of particles capable of emitting light when irradiated with light with a preset threshold value. When two or more light receiving means are installed, it is possible to suppress the occurrence of a count error by adopting data indicating the maximum value among the brightness values or area values obtained from each light receiving means (particles). If the overlap occurs, both the brightness value and the area value will be reduced). Further, when a plurality of two-dimensional light receiving elements are installed, the number of particles may be determined by an algorithm for estimating the number of particles based on the obtained plurality of shape data.

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 can be arranged at a position where light can be received, and can be appropriately selected according to the purpose, but the angle formed by each light receiving direction is different. It is preferably arranged at a position that does not reach 0 °. 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 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 two 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 the adjacent light receiving means is 360 ° by the number of light receiving means, etc. It is preferable to make the angle divided. 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 as to be positioned so that the light receiving directions of the adjacent light receiving means are each 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 a plurality of droplets. As a result, the plurality of droplets ejected from different positions are irradiated with light from the light irradiating means, and the light emitted from the particles can be received more reliably.
Here, synchronization means, for example, at the timing when a plurality of droplets are ejected and the droplets are irradiated with light when they reach a predetermined position, and when the light is irradiated, the particles capable of emitting light are emitted. , Means that the light receiving means receives light emission. That is, the light receiving means detects light emission with a delay of a predetermined time with respect to the ejection of a plurality of droplets from different positions and the irradiation of light by the light irradiating means.

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 plurality of droplets based on the light emitted by the light receiving means.
The particle counting step is a step of counting particles contained in a plurality of droplets based on the light emitted by the 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 if the particles do not transmit light, in order to obtain the brightness value of the light emission from two or more light receiving means, the particles are counted based on the brightness value of the light emission in which the light emission does not overlap or the light emission does not overlap. Can be done.

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. The process of counting the number of light emission within the range, (5) the process of counting the number of inflection points on the outer circumference of the shape on the light receiving surface of light emission as the number of particles, and (6) the process of counting the number of inflection points 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 of (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 that the light emission having the radius of curvature smaller than the predetermined range is excluded. 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 the 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 on the shape of the light emitting surface, and counts the number of centers of the circle as the number of particles when the radius of curvature within a predetermined range is calculated. 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.
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 based on the two-dimensional image obtained from the light receiving means.
Examples of software that performs image processing include ImageJ (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.

When the particle counting means determines that the number of particles is 0, it is preferable to further eject the droplets. As a result, a predetermined number of particles can be attached to the object to be adhered.
It is also possible to prevent the droplets having 0 particles from adhering to the adherend. As a result, it is possible to prevent the adherend from being soiled.

Further, when the particle counting means determines that the number of particles is one or more, the droplets determined to have the number of particles of one or more are attached to the adherend and then adhered to the adherend. It is also possible to move to the next step such as the step of ejecting the droplets to a position different from the position where the particles are made.
Further, after ejecting the droplets a predetermined number of times from different positions, the luminance value of the emission and the shape information on the light receiving surface of the emission recorded in the recording means described later are read out, and the particles contained in each of the ejected droplets. You may try to count the number of.

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.

<Other means and other processes>
The other means are not particularly limited and may be appropriately selected depending on the intended purpose, but it is preferable to have an optical system and recording means. 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. Examples thereof include a filter for facilitating the reception of light emission.

The recording means is not particularly limited as long as it can record 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 depending on the purpose. For example, RAM (Random Access Memory) ) And so on.

(Drop formation device)
The droplet forming apparatus of the present invention includes the particle counting apparatus of the present invention, a droplet ejection means, and if necessary, other means.

<Drop ejection means and droplet ejection process>
The droplet ejection means is a means having a plurality of ejection ports as a ejection start point.
The plurality of discharge ports correspond to different positions (discharge start points) at which a plurality of droplets are discharged in the light irradiation means of the particle counting device of the present invention.

The operation method of the droplet ejection means is not particularly limited and can be appropriately selected according to the purpose. For example, a piezoelectric pressurization method using a piezoelectric element, a thermal method using a heater, or a liquid by electrostatic attraction. An inkjet head or the like by an electrostatic method or the like that pulls a squeeze can be mentioned. Among these, the piezoelectric pressurization method is preferable because the heat and electric field damage to the particles are relatively small.

The droplet ejection means preferably has a liquid holding portion, a film-like member, and a vibrating member, and more preferably has other members as needed.
The droplet ejection means may be either an open head or a closed head.

-Liquid holder-
The liquid holding portion is a portion that holds a liquid containing particles that can emit light when irradiated with light.
When the droplet ejection means is an open head, it has an open portion to the atmosphere on the upper side. The position of the open part to the atmosphere is not limited to the upper part. Bubbles mixed in the liquid are configured so that they can be discharged from the open part to the atmosphere.

The shape, size, material, and structure of the liquid holding portion are not particularly limited and can be appropriately selected depending on the intended purpose.
Examples of the material of the liquid holding portion include stainless steel, nickel, aluminum and the like, silicon dioxide, alumina, zirconia and the like.
Among these, when using cells or proteins as particles, it is preferable to use a material having low adhesion to cells or proteins.
It is generally said that the cell adhesion depends on the contact angle of the material with water, and when the material has high hydrophilicity or hydrophobicity, the cell adhesion is low. Various metal materials and ceramics (metal oxides) can be used as the material having high hydrophilicity, and fluororesin or the like can be used as the material having high hydrophobicity.
In addition to these, it is also conceivable to reduce cell adhesion by coating the surface of the material. For example, the surface of the material can be coated with the above-mentioned metal or metal oxide material, or with a synthetic phospholipid polymer imitating a cell membrane (for example, Lipidure manufactured by NOF CORPORATION).

-Membrane-like member-
The film-like member is a member in which a plurality of discharge ports (nozzles) are formed, and the liquid held in the liquid holding portion is discharged as droplets from the plurality of discharge ports by vibration due to the amplitude motion thereof.
The film-like member is fixed to the lower end of the liquid holding portion when the droplet ejection means is an open head.
The film-like member is fixed to the upper end portion of the liquid holding portion when the droplet ejection means is a closed head.
The liquid held in the liquid holding portion is discharged as droplets from a plurality of discharge ports which are through holes by vibration of the film-like member.

The planar shape, size, material, and structure of the film-like member are not particularly limited and may be appropriately selected depending on the intended purpose.
Examples of the planar shape of the film-like member include a circle, an ellipse, a rectangle, a square, and a rhombus.
As the material of the film-like member, if it is too soft, the film-like member easily vibrates, and it is difficult to immediately suppress the vibration when it is not discharged. Therefore, it is preferable to use a material having a certain degree of hardness, for example. Examples thereof include metals, ceramics, and polymer materials, and specific examples thereof include stainless steel, nickel, aluminum, silicon dioxide, alumina, and zirconia. Among these, when cells or proteins are used as particles as in the case of the liquid holding portion, it is preferable to use a material having low adhesion to the cells or proteins.

-Multiple outlets-
The plurality of discharge ports are not particularly limited in terms of the number of arrangements, arrangement mode, spacing (pitch), opening shape, opening size, and the like, and can be appropriately selected depending on the intended purpose.
The number of discharge ports arranged is not particularly limited and may be appropriately selected depending on the intended purpose, but it is preferable that one or more rows are arranged along the length direction of the discharge surface of the droplet discharge means. More preferably, 1 row or more and 4 rows or less. By providing one or more rows of ejection ports, the number of droplets ejected per unit time can be increased, and the rows can be changed according to the type of particles (for example, cell type) to eject at one time. can.
The number of discharge ports per row is not particularly limited and is appropriately selected according to the purpose, but is preferably 2 or more and 100 or less, more preferably 2 or more and 50 or less, and 2 or more and 12 pieces. The following is more preferable. When the number of ejection ports per row is 2 or more and 100 or less, it is possible to provide a highly productive particle counting device capable of increasing the number of droplets ejected per unit time.
The arrangement mode of the plurality of discharge ports is not particularly limited and may be appropriately selected depending on the intended purpose, and may be a regular arrangement (for example, a houndstooth arrangement) or an irregular arrangement. ..
When a plurality of discharge ports are in a plurality of rows, a partition member should be provided between the rows in order to prevent interference between droplets discharged from adjacent discharge ports and improve the detection sensitivity of particles. Is preferable. Examples of the partition member include a partition plate and the like.
The plurality of discharge ports are preferably arranged side by side at equal intervals, and the interval (pitch) P, which is the shortest distance between the centers of adjacent discharge ports, is not particularly limited and may be appropriately selected according to the purpose. However, for example, it is preferably 50 μm or more and 1,000 μm or less.
The opening shape of the plurality of discharge ports is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a circular shape, an elliptical shape, and a quadrangular shape.
The average diameter of the plurality of discharge ports is not particularly limited and can be appropriately selected depending on the purpose, but in order to prevent the particles from clogging the discharge ports, the average diameter may be at least twice the size of the particles. preferable.
When the particles are, for example, animal cells, particularly human cells, the size of the human cells is generally 5 μm or more and 50 μm or less, so that the average diameter of the plurality of outlets is adjusted to the cells to be used. It is preferably 10 μm or more and 100 μm or less.
On the other hand, if the droplets become too large, it becomes difficult to achieve the purpose of forming fine droplets, so that the average diameter of the plurality of ejection ports is preferably 200 μm or less. Therefore, the average diameter of the plurality of discharge ports is more preferably 10 μm or more and 200 μm or less.

-Vibration member-
The vibrating member is a member that vibrates a film-like member to eject droplets from a plurality of ejection ports (nozzles).
The vibrating member is formed on the lower surface side of the film-like member when the droplet ejection means is an open head.
The vibrating member is formed on the upper surface side of the film-like member when the droplet ejection means is a closed head.
The shape, size, material, and structure of the vibrating member are not particularly limited and can be appropriately selected according to the purpose.
The shape of the vibrating member is not particularly limited and can be appropriately designed according to the shape of the film-like member. For example, when the planar shape of the film-like member is circular, the case of a closed head is used. , It is preferable to provide a circular vibrating member. Further, in the case of an open head, it is preferable to form a vibrating member having an annular shape (ring shape) in a planar shape around the plurality of discharge ports.

A piezoelectric element is preferably used as the vibrating member. The piezoelectric element may have, for example, a structure in which electrodes for applying a voltage are provided on the upper surface and the lower surface of the piezoelectric material. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means, compressive stress is applied in the lateral direction of the film surface, and the film-like member can be vibrated in the vertical direction of the film surface.
The piezoelectric material is not particularly limited and may be appropriately selected depending on the intended purpose. For example, lead zirconate titanate (PZT), bismuth iron oxide, metal niobate, barium titanate, or materials thereof. For example, a metal or a different oxide is added to the mixture. Among these, lead zirconate titiate (PZT) is preferable.

<Other means>
As other means, for example, it is preferable to have a driving means.
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.

Here, an embodiment of the droplet forming apparatus of the present invention will be described in detail with reference to the drawings.
Since the droplet forming apparatus of the present invention corresponds to the particle counting apparatus of the present invention provided with the droplet ejection means and is included in the droplet forming apparatus of the present invention, the following embodiment of the droplet forming apparatus of the present invention An embodiment of the particle counting device of the present invention will also be described through the description of the above.
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.

<First Embodiment>
FIG. 1 is a schematic view showing an example of the droplet forming apparatus of the first embodiment.
The droplet forming apparatus 1 of FIG. 1 includes a droplet ejection means 10, a driving means 20, a light irradiation means 30, a light receiving means 41, and a particle counting means 50.
FIG. 1 typically shows a state in which one droplet is ejected from one of the four ejection ports 14, but the timing of ejecting the droplet is appropriately adjusted and the four ejection ports are ejected. Four droplets 210 can be continuously ejected from the outlet 14.
In FIG. 1, only one light receiving means 41 is represented, but an arbitrary position capable of receiving light emitted from particles contained in the four droplets discharged from the four ejection ports 14 and an arbitrary position and Can be arranged in numbers.

The droplet ejection means 10 is a closed head in the present embodiment, and particles capable of emitting light when a liquid holding portion 11 and four ejection ports 14 are formed and irradiated with light held by the liquid holding portion 11. It has a film-like member 12 that discharges four droplets 210 in the discharge direction shown by D3 in FIG. 1 from four discharge ports 14 by vibrating the vibrating member 13 to discharge the liquid 201 containing 200.
The liquid holding portion 11 may be divided into four liquid chambers for each of the four discharge ports 14 by a partition wall.

As the operation method of the droplet ejection means 10, a piezoelectric pressurization method using a piezoelectric element is used.

The liquid holding portion 11 is a portion that holds the liquid 201 containing the particles 200, and is formed of, for example, stainless steel, a metal such as nickel or aluminum, silicon, or ceramics.
Although four discharge ports 14 which are through holes are provided on the lower surface side of the liquid holding portion 11, the number of discharge ports is not limited to four. The four ejection ports 14 are arranged in a row, and the four ejected droplets 210 can be irradiated with the light L from the light irradiating means 30.

The diameter of the four discharge ports 14 is preferably twice or more the size of the particles 200, and more preferably 10 μm or more and 100 μm or less.

Here, the arrangement state of the plurality of discharge ports 14 on the discharge surface of the droplet discharge means will be described.
FIG. 7 is a schematic plan view showing an example of the arrangement state of the discharge ports 14 on the discharge surface 11a of the droplet discharge means 10, and six discharge ports 14 are arranged in a row along the length direction of the discharge surface 11a. It is installed.
FIG. 8 is a schematic plan view showing an example of the arrangement state of the discharge ports 14 on the discharge surface 11a of the droplet discharge means 10, and 12 discharge ports 14 are arranged in a row along the length direction of the discharge surface 11a. It is installed.
FIG. 9 is a schematic plan view showing an example of the arrangement state of the discharge ports 14 on the discharge surface 11a of the droplet discharge means 10, and a total of 36 discharge ports 14 are arranged in three rows along the length direction of the discharge surface 11a. It is arranged.
FIG. 10 is a schematic plan view showing an example of the arrangement state of the discharge ports 14 on the discharge surface 11a of the droplet discharge means 10, and a total of 20 discharge ports 14 are arranged in three rows along the length direction of the discharge surface 11a. It is arranged (so-called houndstooth arrangement).
FIG. 11 is a schematic plan view showing an example of the arrangement state of the discharge ports 14 on the discharge surface 11a of the droplet discharge means 10, and a total of 36 discharge ports 14 are arranged in three rows along the length direction of the discharge surface 11a. It is arranged, and a partition member 25 is provided between each row.

The film-like member 12 is a portion in which four discharge ports 14 are formed, and four droplets 210 are discharged from the four discharge ports 14 by vibrating the liquid 201 containing the particles 200 held in the liquid holding portion 11. In the present embodiment, since the droplet ejection means 10 is a closed head, it is fixed to the upper end portion of the liquid holding portion 11. The planar shape of the film-like member 12 is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a circular shape, an elliptical shape, and a quadrangular shape.

In the present embodiment, the vibrating member 13 is arranged on the upper surface of the film-like member 12 because the droplet ejection means 10 is a closed head. By supplying a drive signal from the drive means 20 to the vibrating member 13, the film-like member 12 can be vibrated. Thereby, four droplets 210 can be ejected from the four ejection ports 14.
The shape of the vibrating member 13 can be designed according to the shape of the film-like member 12. For example, when the planar shape of the film-like member is circular, it is preferable to provide a circular vibrating member.
A piezoelectric element is used as the vibrating member 13, and lead zirconate titanate (PZT) is used as the piezoelectric element.

The light irradiating means 30 irradiates the four flying droplets 210 with light. The light irradiation means 30 can emit light in synchronization with the ejection of droplets by the droplet ejection means 10 (synchronous with the drive signal supplied from the drive means 20 to the droplet ejection means 10). The term “flying” refers to a state in which the droplet 210 is ejected from the droplet ejecting means 10 until the droplet is deposited on the object to be adhered.
Here, synchronization does not mean that light is emitted at the same time as the droplet ejection means 10 ejects the droplet (at the same time that the driving means 20 supplies the driving signal to the droplet ejection means 10), but the droplets fly. This means that the light irradiating means 30 emits light at the timing when the droplets are irradiated with light when the predetermined position is reached. That is, the light irradiating means 30 emits light with a predetermined time delay with respect to the ejection of the four droplets 210 by the droplet ejection means 10 (the drive signal supplied from the driving means 20 to the droplet ejection means 10). ..

The light L emitted from the light irradiation means 30 preferably irradiates one flying droplet. When four droplets 210 are simultaneously ejected from the four ejection ports 14 and the four droplets 210 are simultaneously irradiated with light L, the light emitted from the particles 200 contained in the four droplets is simultaneously received. Therefore, it becomes difficult to count the number of particles 200 contained in each droplet. That is, in order to improve the accuracy of counting the number of particles in the droplet, it is preferable to shift the timing at which the four droplets are ejected from the four ejection ports 14.

The light L emitted from the light irradiating means 30 is preferably pulsed light, and for example, a solid-state laser, a semiconductor laser, a dye laser, or the like is preferably used. When the light is pulsed light, the pulse width is preferably 10 μs or less, more preferably 1 μs or less. The energy per unit pulse largely depends on the optical system such as the presence or absence of light collection, but is preferably 0.1 μJ or more, and more preferably 1 μJ or more.

The light receiving means 41 receives the fluorescence emitted by the particles 200 absorbing the light as excitation light when the particles 200 are contained in the four flying droplets. Since the fluorescence is emitted from the particles 200 in all directions, the light receiving means 41 can be arranged at an arbitrary position where the fluorescence can be received. At this time, in order to improve the contrast, it is preferable to arrange the light receiving means 41 at a position where the light emitted from the light irradiating means 30 is not directly incident.
Examples of the light receiving means 41 include one-dimensional elements such as a photodiode and a photosensor, but when highly sensitive measurement is required, it is preferable to use a photomultiplier tube or an avalanche photodiode. As the light receiving means, for example, a two-dimensional element such as a CCD (Charge Coupled Device), a CMOS (Complementary Metal Oxide Semiconductor), or a gate CCD may be used.

Since the light emitted from the particles 200 is weaker than the light emitted by the light irradiating means 30, a filter for attenuating the wavelength range of light may be installed in front of the light receiving means 41 (light receiving surface side). As a result, in the light receiving means 41, light having a very high contrast can obtain an image of the particles 200. As the filter, for example, a notch filter that attenuates a specific wavelength region including a wavelength of light can be used.

The particle counting means 50 detects the number of particles 200 (including the case where it is zero) in four droplets based on the information from the light receiving means 41.
The particle counting means 50 may include, for example, a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), a main memory, and the like. In this case, various functions of the particle counting means 50 can be realized by reading the program recorded in the ROM or the like into the main memory and executing the program by the CPU. However, a part or all of the particle counting means 50 may be realized only by hardware. Further, the particle counting means 50 may be physically composed of a plurality of devices or the like.

The particle counting means 50 can detect the number of particles 200 by comparing, for example, the amount of light received by the light receiving means 41 with a preset threshold value. In this case, a one-dimensional element or a two-dimensional element may be used as the light receiving means 41.
When a two-dimensional element is used as the light receiving means 41, the particle counting means 50 performs image processing for calculating the brightness value or area of the particles 200 based on the two-dimensional image obtained from the light receiving means 41. You may use it. In this case, the particle counting means 50 calculates the brightness value or area value of the particles 200 by image processing, and compares the calculated brightness value or area value with a preset threshold value to obtain the number of particles 200. Can be detected. In addition, when a two-dimensional element is used, non-ejection detection is possible by taking an image of the droplet at the timing immediately before receiving the light emission.

As the particle 200, cells are preferable, and at least one of cells stained with a fluorescent dye and cells capable of expressing a fluorescent protein is more preferable. In these, the autofluorescent means 41 receives the autofluorescence, and the particle counting means 50 can detect the number of cells contained in the droplet.
Examples of the fluorescent dye include cell tracker orange and cell tracker red.
Examples of the fluorescent protein include a green fluorescent protein (GFP; green fluorescent protein), a red fluorescent protein (RFP; Red Fluorescent Protein), and a yellow fluorescent protein (YFP; Yellow Fluorescent Protein).

<Modification 1 of the first embodiment>
FIG. 2 is a schematic view showing an example of the droplet forming apparatus of the first modification of the first embodiment. In the first modification of the first embodiment, the same components as those of the above-described embodiment are designated by the same reference numerals, and the description thereof will be omitted.

The difference between the droplet forming apparatus 1A shown in FIG. 2 and the droplet forming apparatus 1 of the first embodiment shown in FIG. 1 is that four ejections are made through the first lens 52 and the second lens 53. Even when droplets are ejected from the outlet 14 at the same time and light is simultaneously applied to the four droplets 210, the number of particles contained in each of the four droplets can be counted. The number of discharge ports is not limited to four.
In FIG. 2, the positions of the first lens 52, the second lens 53, and the particle counting means 50 are examples, and are in the vicinity of the four droplets 210 ejected from the four ejection ports 14 and are liquids. There is no particular limitation as long as the position does not interfere with the dropping of drops or the dropping of drops on the object to be adhered. Further, in FIG. 2, the position of the light receiving means 41 is an arbitrary position capable of receiving light emitted from the particles 200 contained in the four droplets 210 ejected from the four ejection ports 14, and is covered with the droplets. It can be placed in a position that does not interfere with the drip on the object to be worn.

The light receiving means 41 has a structure in which a plurality of light receiving means are formed in an array. When each of the four droplets is irradiated with light, the light emitting Lf1 from the particles 200 is redirected by the first lens 52, collected by the second lens 53, and received by the second lens 53. It is possible to form an image on one light receiving means constituting 41. Thereby, even when light is irradiated to the four droplets at the same time, the number of particles 200 contained in each of the four droplets can be counted.
If necessary, the first and second lenses may be combined with a mirror for adjustment.

As the first lens 42 and the second lens 43, it is preferable to use a lens having a high numerical aperture (NA), and in particular, the objective lens and the fθ lens inject light perpendicularly to the light receiving means 41. This is preferable because it is possible to make the light receiving means 41 less dependent on the location of the light receiving means 41. Here, the objective lens is a lens capable of obtaining a magnified image with less aberration. The fθ lens is a lens in which y = fθ is established when the image height is y, the focal length is f, and the angle of light incident on the lens is θ.
As the light receiving means 41, for example, a CCD array, a CMOS array, a photodiode array, or the like can be used. Of these, it is most preferable to use a photodiode array from the viewpoint of sensitivity and time response.

<Modification 2 of the first embodiment>
FIG. 3 is a schematic view showing an example of the droplet forming apparatus of the second modification of the first embodiment. In the second modification of the first embodiment, the same reference numerals are given to the same configurations as those of the above-described embodiment, and the description thereof will be omitted.

The difference between the droplet forming apparatus 1B shown in FIG. 3 and the droplet forming apparatus 1 of the first embodiment shown in FIG. 1 is the particles contained in the four droplets 210 ejected from the four ejection ports 14. The point is that the two light receiving means 41 and 42 receive light from different directions. The number of discharge ports is not limited to four.
The droplet forming apparatus 1B includes a droplet ejection means 10, a driving means 20, a light irradiating means 30, light receiving means 41 and 42, and a particle counting means 50.
In FIG. 3, the positions and numbers of the light receiving means 41 and 42 are examples, and any position and an arbitrary position capable of receiving light emitted from the particles 200 contained in the four droplets 210 discharged from the four ejection ports 14 and Can be arranged in numbers.

The droplet ejection means 10 is a closed head, contains the liquid 201 containing the particles 200 when the liquid holding portion 11 is irradiated with light, and is caused by the vibration of the vibrating member 13 arranged above the liquid holding portion 11. , The four droplets 210 containing the particles 200 can be ejected from the four ejection ports 14 in the ejection direction shown by D3 in FIG.

The driving means 20 is electrically connected to the vibrating member 13 of the droplet discharging means 10, and applies a driving voltage to the vibrating member 13 to vibrate the vibrating member 13 to vibrate the four discharging ports 14 to four liquids. Drop 210 is discharged.

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 four droplets 210 with laser light L as light in accordance with the ejection timing of the four droplets 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 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 Lf1 and the brightness value of the light emitting Lf2, and the shape of the light emitting Lf1 on the light receiving surface and the shape of the light emitting Lf2 on the light receiving surface, respectively.

The particle counting means 50 is based on information on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2 received by the two light receiving means 41 and 42, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. The particles 200 contained in one droplet 210 are counted.

FIG. 12 is an explanatory diagram showing an example of the positional relationship between the two light receiving means 41 and 42 in the droplet forming apparatus 1B shown in FIG. In FIG. 12, the ejection direction of the droplet 210 is the depth direction of the paper surface, and the object to be adhered 300 is arranged directly below the ejection port 14. In FIG. 12, one of the four discharge ports is represented as a representative.
As shown in FIG. 12, the light irradiating means 30, the mirror 60, and the lens 70 are liquids in which the laser beam L irradiated by the light irradiating means 30 is reflected by the mirror 60 and discharged in the ejection direction through the lens 70. It is arranged so as to be focused on the droplet (position of the discharge port 14 in FIG. 12).
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. 13 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. 13, 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. 13, the light emission does not overlap in the image P1, but the light emission overlaps in the image P2.
When counting the particles 200 from the image P1 and the image P2 based on the brightness of light emission, either of the above processes (1) or (2) may be performed to count the number of particles 200.
When counting the particles 200 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 particles 200.

14A to 14C 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. 14A to 14C, 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. 14A, 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. 14B, 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. 14C, 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.

15A and 15B 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. 15A and 15B, similarly to FIGS. 14A to 14C, the light and shade of the image indicates the brightness of light emission, and the lighter the color, the higher the brightness of light emission. However, it differs from FIGS. 14A to 14C in that one particle is contained in the droplet and the particle exists near the outer edge of the droplet.
As shown in FIG. 15A, the outer edge of the droplet emits light, and when the number of particles is determined 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. 15B, when the number of particles is determined based on the luminance value of light emission as in the case of FIG. 15A, it is difficult to determine whether the number of particles is one or two. 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.

16A to 16C are images 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. 16A to 16C show SPHERO Fluorescent Nile Red droplets (manufactured by Bay Biosciences Co., Ltd., 1% (w / v), diameter 10 μm to 14 μm) as particles, ion-exchanged water as a liquid, and a piezoelectric element. A membrane vibration type inkjet head is driven by a piezoelectric element with a sinusoidal voltage (20,000 Hz, 1 cycle, 4.6 V) to eject droplets containing particles so as to form a sphere with a diameter of 80 μm, which is highly effective as a light receiving means. Set the exposure time of the sensitivity camera (pco.edge, manufactured by Tokyo Instruments Co., Ltd.) to 10 ms, and use the YAG laser (Explorer ONE-532-200-KE, Spectra Physics Co., Ltd.) as a means of irradiating the ejected droplets with light. This is an image of a state in which particles are emitted by irradiating a laser beam (100 μJ at 10 kHz) emitted by (manufactured by). 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 liquid to be discharged, the particles, and the like.

In FIG. 16A, 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. 16A, 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. 16B 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. be.
As shown in FIG. 16B, light emitted from particles existing near the outer edge of the droplet may be reflected inside the spherical surface of the droplet.

FIG. 16C 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. 16C, 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. 17 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. 17, 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 light received by the second light receiving means are received. The number of particles can be determined based on the brightness value 2 of the emitted light.
Further, as shown in FIGS. 16A to 16C, 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 number of particles is 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 is recorded in a recording means or the like.

Next, using the droplet forming apparatus of the present invention, the flow of counting the particles contained in the plurality of droplets ejected from the droplet ejection means is according to the step represented by S in the flowchart of the flowchart shown in FIG. This will be described with reference to FIG. 3 and the like.
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 particles 200 from the ejection port (nozzle) 14 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 50 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 are emitted 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 Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. After that, the particle counting means 50 shifts the processing to S103.

In S103, the particle counting means 50 receives information on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface, which are received by the two light receiving means 41 and 42. Based on the above, the particles 200 contained in the droplet 210 are counted using the calibration curve shown in FIG. 17, and this process is completed.

Next, using the droplet forming apparatus of the present invention, the flow of counting the particles contained in the plurality of droplets ejected from the droplet ejection means is according to the step represented by S in the flowchart of the flowchart shown in FIG. This will be described with reference to FIG. 3 and the like.
This flowchart is a procedure used, for example, when the number of particles 200 contained in the droplet 210 to be 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 particles 200, the brightness value of the light emitting Lf1 and the light emitting Lf2 from the particles 200, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface each time the particles are discharged. This is a procedure for counting the particles 200 based only on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2.

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 particles 200 from the ejection port 14 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 50 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 are emitted 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 Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. After that, the particle counting means 50 shifts the processing to S203.

In S203, the particle counting means 50 uses the calibration curve shown in FIG. 17 based on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2 received by the two light receiving means 41 and 42, and the particles contained in the droplet 210. Count 200. After that, the particle counting means 50 shifts the processing to S204.

In S204, the particle counting means 50 that counts the particles 200 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 50 determines that the droplet discharging means 10 has discharged the droplet 210 a predetermined number of times, the particle counting means 50 ends this process. When the particle counting means 50 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 droplet forming apparatus of the present invention, the flow of counting the particles contained in the plurality of droplets ejected from the droplet ejection means is set in the step represented by S in FIG. 20 of the flowchart shown in FIG. Therefore, it will be described with reference to FIG. 3 and the like.
This flowchart is a procedure used, for example, when it is desired to continuously eject the droplet 210 by the droplet ejection means 10 and then check how much the particles 200 are contained in the continuously ejected droplet 210. be.

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 particles 200 from the ejection port 14 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 50 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 are emitted 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 Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. The particle counting means 50 records information on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface and the shape of the light emitting Lf2 on the light receiving surface in a recording means (not shown) for each droplet 210. The process shifts to S303.

In S303, the particle counting means 50 that counts the particles 200 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 50 determines that the droplet 210 has been ejected a predetermined number of times by the droplet ejection means 10, the process shifts to S304. When the particle counting means 50 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 50 that determines that the droplet 210 has been ejected a predetermined number of times has the luminance value of the emission Lf1 and the luminance value of the emission Lf2 recorded in the recording means, the shape of the emission Lf1 on the light receiving surface, and the emission Lf2. Based on the shape information on the light receiving surface, the particles 200 are counted for each droplet 210 using the calibration curve shown in FIG. 17, and this process is completed.

Next, using the droplet forming apparatus of the present invention, the flow of counting the particles contained in the plurality of droplets ejected from the droplet ejection means is according to the step represented by S in the flowchart of the flowchart shown in FIG. This will be described with reference to FIG. 36 and the like.
In this flowchart, when it is determined that the number of particles 200 cannot be counted based only on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2, the particles are based on the shape of the light receiving surface of the light emitting Lf1 and the shape of the light receiving surface of the light emitting Lf2. This is a procedure for determining the number of 200 particles.

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 particles 200 from the ejection port 14 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 50 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 are emitted 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 Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. After that, the particle counting means 50 shifts the processing to S403.

In S403, the particle counting means 50 uses the calibration curve shown in FIG. 17 to measure the particles 200 for each droplet 210 based on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2 received by the two light receiving means 41 and 42. Determine the number of. At this time, when the particle counting means 50 determines that the brightness value of the received light emitting Lf2 is not included in the undeterminable region shown in FIG. 17, the process shifts to S404. When the particle counting means 50 determines that it is included in the undeterminable region, the process shifts to S405.

In S404, the particle counting means 50 determined not to be included in the undeterminable region is the calibration curve shown in FIG. 17 based on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2 received by the two light receiving means 41 and 42. The number of particles 200 contained in the droplet 210 is counted using the above, and this process is completed.

In S405, the particle counting means 50 determined to be included in the undeterminable region has information on the shape of the light emitting Lf1 received by the two light receiving means 41 and 42 on the light receiving surface and the shape information of the light emitting Lf2 on the light receiving surface, that is, shape data. The radius of curvature is calculated based on.
Next, the particle counting means 50 determines whether or not the obtained radius of curvature is within a predetermined range. When the particle counting means 50 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 50 that determines that all the radii of curvature are within a predetermined range counts the number of centers of the obtained radii of curvature as the number of particles 200 contained in the droplet 210, and ends this process. do.

Next, using the droplet forming apparatus of the present invention, the flow of counting the particles contained in the plurality of droplets ejected from the droplet ejection means is according to the step represented by S in the flowchart of the flowchart shown in FIG. This will be described with reference to FIG. 3 and the like.

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 particles 200 from the ejection port 14 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 50 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 are emitted 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 Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. After that, the particle counting means 50 shifts the processing to S503.

In S503, the particle counting means 50 uses the calibration curve shown in FIG. 17 based on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2 received by the two light receiving means 41 and 42, and the particles contained in the droplet 210. Count 200. After that, the particle counting means 50 shifts the processing to S504.

In S504, the particle counting means 50 determines whether or not the number of counted particles 200 is 0. When the particle counting means 50 determines that the number of counted particles 200 is 0, the process shifts to S501. When the particle counting means 50 determines that the number of counted particles 200 is not 0, the particle counting means 50 ends this process.

Next, when the particle counting means determines that the number of particles contained in the plurality of ejected droplets is 0 by using the droplet forming apparatus of the present invention, the droplet ejection means ejects the droplets again. The flow of repeating the process until the particles 200 are counted will be described with reference to FIG. 3 and the like according to the steps 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 particles 200 from the ejection port 14 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 50 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 Lf1 and the light emitting Lf2 at the timing when the particles 200 are emitted 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 Lf1 and the brightness value of the light emitting Lf2, the shape of the light emitting Lf1 on the light receiving surface, and the shape of the light emitting Lf2 on the light receiving surface. After that, the particle counting means 50 shifts the processing to S603.

In S603, the particle counting means 50 uses the calibration curve shown in FIG. 17 based on the brightness value of the light emitting Lf1 and the brightness value of the light emitting Lf2 received by the two light receiving means 41 and 42, and the particles contained in the droplet 210. Count 200. After that, the particle counting means 50 shifts the processing to S604.

In S604, the particle counting means 50 that counts the particles 200 determines whether or not the counted particles 200 are 0. When the particle counting means 50 determines that the number of counted particles 200 is 0, the process shifts to S605. When the particle counting means 50 determines that the number of counted particles 200 is not 0, the particle counting means 50 ends this process.

In S605, the particle counting means 50 discharges the droplet 210 determined to have 0 particles 200 so as not to adhere to the adherend, and returns the process to S601.

<Second embodiment>
FIG. 4 is a schematic view showing an example of the droplet forming apparatus of the second embodiment. In the second embodiment, the same components as those in the above-described embodiment are designated by the same reference numerals, and the description thereof will be omitted.

The difference between the droplet forming apparatus 1C shown in FIG. 4 and the droplet forming apparatus 1 of the first embodiment shown in FIG. 1 is that the droplet ejection means 10 is changed to an open head.
The droplet forming device 1C of FIG. 4 has a droplet ejection means 10, a driving means 20, a light irradiating means 30, a light receiving means 41, and a particle counting means 50.
FIG. 4 typically shows a state in which one droplet 210 is ejected from one of the three ejection ports 14, but three droplets are ejected by appropriately adjusting the timing of ejecting the three droplets. It can be continuously discharged from the discharge port 14. The number of discharge ports is not limited to three.
In FIG. 4, the position and number of the light receiving means 41 are an example, and the light emission from the particles contained in the three droplets 210 discharged from the three ejection ports 14 is arranged at an arbitrary position and number capable of receiving light. can do.

The droplet ejection means 10 is an open head in the present embodiment, and the liquid holding portion 11 and the three ejection ports 14 are formed, and the liquid 201 held in the liquid holding portion 11 is vibrated by the vibrating member 13. It has a film-like member 5 that discharges as three droplets 210 from the discharge port 14.
The liquid holding portion 11 is a portion that holds the liquid 201 containing the particles 200 capable of emitting light when irradiated with light, and since it is an open head in the present embodiment, it has an atmospheric opening portion 15 at the upper portion. .. By this, the bubbles mixed in the liquid 201 can be discharged from the open portion to the atmosphere.

In the present embodiment, the vibrating member 13 is arranged on the lower surface of the film-like member 12 because the droplet ejection means 10 is an open head. The shape of the vibrating member 13 can be designed according to the shape of the film-like member 12. For example, when the planar shape of the film-shaped member 12 is circular, it is preferable to form the vibrating member 13 having an annular (ring-shaped) planar shape around the three discharge ports 14.
By supplying a drive signal from the drive means 20 to the vibrating member 13, the film-like member 12 can be vibrated. Thereby, three droplets 210 can be ejected from the three ejection ports 14.

In the present embodiment, since the droplets are formed by the inertia of the vibration of the film-like member 12, even a particle suspension liquid having a high surface tension (high viscosity) can be discharged.
As the material of the film-like member 12, if it is too soft, the film-like member 12 easily vibrates, and it is difficult to immediately suppress the vibration when the film-like member 12 is not discharged. Therefore, it is preferable to use a material having a certain degree of hardness. As the material of the film-like member 12, for example, a metal material, a ceramic material, a polymer material having a certain degree of hardness, or the like can be used.
As the material of the liquid holding portion 11 and the film-like member 12, when cells are used as particles, a material having low adhesion to cells or proteins can be used.

The driving means 20 selectively (for example, for example, a discharge waveform that vibrates the film-like member 12 to form a droplet and a stirring waveform that vibrates the film-like member 12 within a range that does not form a droplet) to the vibrating member 13. Can be given (alternately).
That is, the driving means 20 adds the discharge waveform to the vibrating member 13 and controls the vibrating state of the film-like member 12, so that the liquid 201 containing the particles 200 held by the liquid holding portion 11 is discharged from the three discharge ports 14. It can be ejected as three droplets. Further, the driving means 20 can agitate the liquid 201 containing the particles 200 held by the liquid holding portion 11 by adding the stirring waveform to the vibrating member 13 and controlling the vibrating state of the film-like member 12. During stirring, the droplets are not discharged from the three discharge ports 14.

In this way, by stirring the liquid 201 containing the particles 200 held in the liquid holding portion 11 while the droplets are not formed, the particles 200 are prevented from settling and aggregating on the film-like member 12. , The particles 200 can be evenly dispersed in the liquid 201. This makes it possible to suppress clogging of the discharge port and variation in the number of particles 200 in the droplet to be discharged. As a result, the liquid 201 containing the particles 200 can be continuously and stably discharged as droplets for a long time.

In the droplet forming apparatus 1C, air bubbles may be mixed in the liquid 201. Even in this case, in the present embodiment, since the air opening portion 15 is provided above the liquid holding portion 11, the bubbles mixed in the liquid 201 can be discharged to the outside air through the air opening portion 15. This makes it possible to continuously and stably form droplets without discarding a large amount of liquid for discharging bubbles. That is, when air bubbles are mixed in the vicinity of the three discharge ports 14 or when a large number of air bubbles are mixed in the film-like member 12, the discharge state is affected, so that the droplets can be stably formed for a long time. In order to do so, it is necessary to discharge the mixed air bubbles. Normally, the air bubbles mixed on the film-like member 12 move upward naturally or by the vibration of the film-like member 12, but since the liquid holding portion 11 is provided with the atmosphere opening portion 15, the mixed air bubbles can be removed. It can be discharged from the air opening section 15. Therefore, even if air bubbles are mixed in the liquid holding portion 11, it is possible to prevent non-ejection from occurring, and it is possible to continuously and stably form droplets.
The film-like member 12 may be vibrated at a timing when the droplets are not formed within a range where the droplets are not formed, and the bubbles may be positively moved above the liquid holding portion 11.

<Modification 1 of the second embodiment>
FIG. 5 is a schematic view showing an example of the droplet forming apparatus of the first modification of the second embodiment. In the first modification of the second embodiment, the same reference numerals are given to the same configurations as those of the above-described embodiment, and the description thereof will be omitted.

The droplet forming device 1D of the modification 1 of the second embodiment shown in FIG. 5 corresponds to the droplet forming device 1A of the modification 1 of the first embodiment shown in FIG. 2, and the droplet ejection means 10 is provided. Since it is common to the droplet forming apparatus 1A of the first embodiment of the modified example 1 except that it is changed to the open head, refer to the description of the second embodiment and the modified example 1 of the first embodiment. , Detailed description is omitted.

<Modification 2 of the second embodiment>
FIG. 6 is a schematic view showing an example of the droplet forming apparatus of the second modification of the second embodiment. In the second modification of the second embodiment, the same components as those of the above-described embodiment are designated by the same reference numerals, and the description thereof will be omitted.

The droplet forming device 1E of the modification 2 of the second embodiment shown in FIG. 6 corresponds to the droplet forming device 1B of the modification 2 of the first embodiment shown in FIG. 3, and the droplet ejection means 10 is provided. Except for the change to the open head, it is common to the droplet forming apparatus 1B of the second embodiment of the first embodiment. Therefore, refer to the description of the second embodiment and the second modification of the first embodiment. , Detailed description is omitted.

Since the droplet forming apparatus of the present invention has improved detection accuracy of particles contained in the ejected droplets and has high productivity capable of increasing the number of droplets ejected per unit time. , But it is particularly preferably used in the dispensing device of the present invention described below.

(Dispensing device)
The dispensing device of the present invention preferably has the above-mentioned droplet forming device of the present invention, an object to be adhered, and a control means, and further has other means as necessary.
When the particle counting device of the droplet forming apparatus determines that the number of particles contained in the droplet is 0, the droplet ejection means may eject the droplet again to the same recess. It is preferable because the particles can be reliably dispensed.

<Object to be adhered>
The object to be adhered is a member in which a plurality of recesses on which droplets ejected from the droplet ejection means of the droplet forming apparatus are deposited are formed.
The material, shape, size, structure, and the like of the ejected droplets are not particularly limited as long as the ejected droplets can be attached to the object to be adhered, and can be appropriately selected according to the purpose.
The material of the object to be adhered is not particularly limited and may be appropriately selected depending on the intended purpose. For example, those formed of semiconductors, ceramics, metals, glass, quartz glass, plastics and the like are preferably mentioned. Be done.
The shape of the object to be adhered is not particularly limited and may be appropriately selected depending on the intended purpose, but for example, a plate shape or a plate shape is preferable.
The structure of the object to be adhered is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may be a single-layer structure or a multi-layer structure.
The number of recesses provided in the object to be adhered is a plurality, preferably two or more, more preferably five or more, and even more preferably 50 or more.

<Control means>
The control means is a means for controlling the relative positional relationship between the droplet ejection means and the plurality of recesses, and includes a CPU (Central Processing Unit) ROM (Read Only Memory), a RAM (Random Access Memory), a main memory, and the like. It has and executes various processes based on the control program for controlling the operation of the entire dispensing device.

<Other means>
The other means are not particularly limited and may be appropriately selected depending on the intended purpose, but it is preferable to have recording means, culturing means, heating means, stirring means, washing means and the like.

The dispensing device of the present invention has high productivity capable of improving the detection accuracy of particles contained in the ejected droplets and increasing the number of ejected droplets per unit time. Since it has an apparatus, it is suitably used for producing an organization that can be widely used in various fields such as regenerative medicine, medicine, cosmetics, and evaluation of safety and efficacy of chemical substances, particularly a three-dimensional organization.

Here, an embodiment of the dispensing device of the present invention will be described in detail with reference to the drawings.

<Third embodiment>
FIG. 24 is a schematic view showing an example of the dispensing device of the third embodiment. In the third embodiment, the droplet forming devices of the first and second embodiments are used as a dispensing device for dispensing particles into the recesses of the adherend. In the third embodiment, the same components as those in the above-described embodiment are designated by the same reference numerals, and the description thereof will be omitted.

The dispensing device 2 shown in FIG. 24 includes a droplet forming device 1, an adherend 300, a stage 400, and a control means 500.
As the droplet forming apparatus 1, the droplet forming apparatus 1 of the first embodiment shown in FIG. 1 is used. In the first embodiment, the same components as those in the above-described embodiment are designated by the same reference numerals, and the description thereof will be omitted.
Instead of the droplet forming apparatus 1, any of the droplet forming apparatus 1A to 1E shown in FIGS. 2 to 6 may be used.

The adherend 300 is arranged on the movable stage 400. The object to be adhered 300 is formed with a plurality of recesses (wells) 310 on which four droplets 210 ejected from the droplet ejection means 10 of the droplet forming apparatus 1 are deposited.
The control means 500 moves the stage 400 to control the relative positional relationship between the droplet ejection means 10 of the droplet forming apparatus 1 and the respective recesses 310. As a result, the droplet 210 containing the particles 200 can be sequentially ejected from the droplet ejection means 10 of the droplet forming apparatus 1 into the respective recesses 310.

The control means 500 can be configured to include, for example, a CPU, a ROM, a RAM, a main memory, and the like. In this case, various functions of the control means 500 can be realized by reading the program recorded in the ROM or the like into the main memory and executing the program by the CPU. However, a part or all of the control means 500 may be realized only by hardware. Further, the control means 500 may be physically composed of a plurality of devices and the like.

FIG. 25 is an example of a flowchart showing the operation of the dispensing device according to the third embodiment. First, in step S101, the droplet ejection means 10 of the droplet forming apparatus 1 ejects the droplet 210 toward the predetermined recess 310.

Next, in step S102, the particle counting means 50 of the droplet forming apparatus 1 detects the number of particles 200 contained in the flying droplet 210, and sends the detection result to the control means 500. If the detection result of the particle counting means 50 is not "one or more" (in the case of zero), the operation of step S101 is repeated.

If the detection result of the particle counting means 50 is "one or more" in step S102, the process proceeds to step S103. In step S103, the control means 500 controls the stage 400 and moves the adherend 300 so that the droplet ejection means 10 of the droplet forming apparatus 1 and the next recess 310 face each other. After that, the process proceeds to step S101 and the same operation is repeated.

As a result, when the number of particles 200 contained in the droplet 210 flying toward the recess 310 is zero, the droplet 210 is ejected again toward the same recess 310, so that the droplet 210 is contained in the plurality of recesses 310. It is possible to reliably dispense the particles 200.

It is also possible to detect the number of particles 200 contained in the flying droplet 210, as shown in FIG. 26, instead of the presence or absence of the particles 200 contained in the flying droplet 210.
FIG. 26 is another example of a flowchart showing the operation of the dispensing device according to the third embodiment.

In FIG. 26, first, the same step S101 as in FIG. 25 is performed, and then in step S202, the particle counting means 50 of the droplet forming apparatus 1 detects the number of particles 200 contained in the flying droplet 210. The detection result is sent to the control means 500. The operation of step S101 is repeated until the detection result of the particle counting means 50 becomes "3".

If the number of particles 200 contained in the droplet 210 increases, the detection accuracy of the particle counting means 50 may decrease. Therefore, the number of particles 200 contained in the droplet 210 discharged at one time is not necessarily 3. It is not necessary to set it to be individual. For example, the number of particles 200 contained in the droplet 210 to be ejected at one time may be set to 0 or 1. In this case, the operation of step S101 is repeated until the total number of particles 200 contained in the droplet 210 is three.

If the detection result of the particle counting means 50 is "3" in step S202, the process proceeds to step S103, and step S103 similar to FIG. 25 is performed. After that, the process proceeds to step S101 and the same operation is repeated. This makes it possible to dispense so that the number of particles 200 in each recess 310 is three.

In the processing of FIGS. 25 and 26, the function of moving the droplet forming apparatus 1 to a predetermined position along the stage 400 can be incorporated into the control means 500 as a program, for example.

Examples of aspects of the present invention are as follows.
<1> A light irradiating means for irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejecting the light from different positions.
A light receiving means that receives light emitted from the particles irradiated with the light, and a light receiving means.
A particle counting means for counting the particles contained in the plurality of droplets based on the light emission received by the light receiving means, and a particle counting means.
It is a particle counting device characterized by having.
<2> The particle counting device according to <1>, which includes two or more light receiving means, and each of the light receiving means receives light emitted from the particles from different directions.
<3> 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.
The particle counting means counts the particles contained in the droplet based on the brightness value of the emission, and the particle counts the droplet based on the shape information on the light receiving surface of the emission. The particle counting device according to <2>, which performs at least one of the second counting processes for counting the contained particles.
<4> The particles according to <3>, 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. It is a counting device.
<5> The particle counting means calculates the radius of curvature based on the shape information of the light emitting surface on the light receiving surface, and the number of centers of the circles when the radius of curvature is calculated within a predetermined range is the number of the particles. The particle counting device according to any one of <3> to <4>, which performs the second counting process for counting as a number.
<6> From <2> to <6>, 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. The particle counting device according to any one of 5>.
<7> The particle counting device according to any one of <2> to <6>, 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.
<8> The particle counting device according to any one of <1> to <7>, wherein the light emitted from the light irradiating means is pulsed light.
<9> The particle counting device according to <8>, wherein the pulse width of the pulsed light is 10 μs or less.
<10> The particle counting device according to any one of <1> to <9>, wherein the particles capable of emitting light when irradiated with the light are cells.
<11> Any of the above <1> to <10>, wherein the particles capable of emitting light when irradiated with the light are at least one of a cell stained with a fluorescent dye and a cell capable of expressing a fluorescent protein. The particle counting device according to the above.
<12> The particle counting device according to any one of <1> to <11>.
As a discharge start point, a droplet discharge means having a plurality of discharge ports and
It is a droplet forming apparatus characterized by comprising.
<13> The droplet forming apparatus according to <12>, wherein the plurality of ejection ports are arranged in one or more rows along the length direction of the ejection surface of the droplet ejection means.
<14> The droplet forming apparatus according to <13>, wherein the number of the plurality of ejection ports is 2 or more and 100 or less per row.
<15> The droplet ejection means
A liquid holding portion that holds a liquid containing particles that can emit light when irradiated with the light, and a liquid holding portion.
A film-like member in which a plurality of discharge ports are formed and the liquid held in the liquid holding portion is discharged as droplets from the plurality of discharge ports by vibration.
The droplet forming apparatus according to any one of <12> to <14>.
<16> The droplet forming apparatus according to <15>, wherein the liquid holding portion has an open portion to the atmosphere.
<17> The droplet forming apparatus according to any one of <15> to <16>, wherein the droplet discharging means has a vibrating member that vibrates the film-like member.
<18> The droplet forming apparatus according to <17>, wherein the vibrating member is a piezoelectric element.
<19> A light irradiation step of irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejecting from different positions with the light.
A light receiving step of receiving light emitted from the particles irradiated with the light, and a light receiving step.
A particle counting step of counting the particles contained in the plurality of droplets based on the light emission received in the light receiving step, and a particle counting step.
It is a particle counting method characterized by containing.
<20> The droplet forming apparatus according to any one of <12> to <18>,
An object to be adhered to which a plurality of recesses on which the droplets ejected from the droplet ejection means are formed are formed.
It is a dispensing device characterized by having.
<21> The dispensing device according to <20>, which has a control means for controlling the relative positional relationship between the droplet ejection means and the plurality of recesses.
<22> When the particle counting device in the droplet forming apparatus determines that the number of particles contained in the droplet is 0,
The dispensing device according to any one of <20> to <21>, wherein the droplet discharging means ejects the droplets again into the same recess.
<23> The dispensing device according to any one of <20> to <22> used for forming an organization.

The particle counting device according to any one of <1> to <11>, the droplet forming device according to any one of <12> to <18>, the particle counting method according to <19>, and the above. The dispensing device according to any one of <20> to <23> can solve the above-mentioned problems in the past and achieve the object of the present invention.

1, 1A, 1B, 1C, 1D, 1E Droplet forming device 2 Dispensing device 10 Droplet ejection means 14 Discharge port 20 Driving means 30 Light irradiation means 41 Light receiving means 42 Light receiving means 50 Particle counting means 200 Particles that can emit light 201 Liquid 210 Droplets L Light Lf Light emission

Japanese Unexamined Patent Publication No. 2008-693

Claims (38)

A light irradiation means that contains particles that can emit light when irradiated with light and that irradiates a plurality of droplets ejected from different positions with the light.
A light receiving means that receives light emitted from the particles irradiated with the light, and a light receiving means.
It has a particle counting means for counting the particles contained in the plurality of droplets based on the light emission received by the light receiving means.
One or more rows of ejection start points of the plurality of droplets are arranged along a direction orthogonal to the ejection direction of the plurality of droplets.
The ejection start points of the plurality of droplets are a plurality of ejection ports provided on the film-like member of the liquid holding portion that holds the liquid containing the particles capable of emitting light when irradiated with the light, and the liquid is A particle counting device characterized in that droplets are discharged from the plurality of discharge ports by vibration of the film-like member. The particle counting device according to claim 1, further comprising two or more light receiving means, each of which receives light emitted from the particles from different directions. 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.
The particle counting means counts the particles contained in the droplet based on the brightness value of the emission, and the particle counts the droplet based on the shape information on the light receiving surface of the emission. The particle counting device according to claim 2, wherein at least one of the second counting processes for counting the contained particles is performed. The particle counting device according to claim 3, wherein when the particle counting means performs the first counting process and determines that the particles cannot be counted, the particle counting means performs the second counting process and counts the particles. The particle counting means calculates the radius of curvature based on the shape information of the light emitting surface on the light receiving surface, and counts the number of centers of the circles within a predetermined range as the number of particles. The particle counting device according to any one of claims 3 to 4, wherein the second counting process is performed. According to any one of claims 2 to 5, 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. The particle counting device according to the description. The particle counting device according to any one of claims 2 to 6, 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 7, wherein the light emitted from the light irradiating means is pulsed light. The particle counting device according to any one of claims 1 to 8, wherein the particles capable of emitting light when irradiated with the light are cells. The particle counting device according to any one of claims 1 to 9, wherein the particles capable of emitting light when irradiated with light are at least one of a cell stained with a fluorescent dye and a cell capable of expressing a fluorescent protein. The particle counting device according to any one of claims 1 to 10.
A droplet forming apparatus comprising: a droplet ejection means having a plurality of ejection ports as an ejection start point. The droplet forming apparatus according to claim 11, wherein the plurality of ejection ports are arranged in one or more rows along the length direction of the ejection surface of the droplet ejection means. The droplet forming apparatus according to claim 12, wherein the number of the plurality of ejection ports is 2 or more and 100 or less per row. The droplet ejection means
A liquid holding portion that holds a liquid containing particles that can emit light when irradiated with the light, and a liquid holding portion.
11. Droplet forming device. The droplet forming apparatus according to claim 14, wherein the liquid holding portion has an open portion to the atmosphere. A light irradiation step of irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejected from different positions with the light.
A light receiving step of receiving light emitted from the particles irradiated with the light, and a light receiving step.
A particle counting step of counting the particles contained in the plurality of droplets based on the light emission received in the light receiving step, and a particle counting step.
Including
One or more rows of ejection start points of the plurality of droplets are arranged along a direction orthogonal to the ejection direction of the plurality of droplets.
The ejection start points of the plurality of droplets are a plurality of ejection ports provided on the film-like member of the liquid holding portion that holds the liquid containing the particles capable of emitting light when irradiated with the light, and the liquid is A particle counting method characterized in that droplets are discharged from the plurality of discharge ports by vibration of the film-like member. The droplet forming apparatus according to any one of claims 11 to 15.
A dispensing device comprising: an adhered object having a plurality of recesses on which the droplets ejected from the droplet ejection means are deposited. The dispensing device according to claim 17, further comprising a control means for controlling the relative positional relationship between the droplet ejection means and the plurality of recesses. When the particle counting device in the droplet forming device determines that the number of particles contained in the droplet is 0,
The dispensing device according to any one of claims 17 to 18, wherein the droplet discharging means discharges the droplet again into the same recess. The dispensing device according to any one of claims 17 to 19, which is used for forming an organization. A light irradiation means that irradiates a plurality of droplets containing particles that can emit light when irradiated with light and that are ejected from different positions with the light.
A light receiving means that receives light emitted from the particles irradiated with the light, and a light receiving means.
It has a particle counting means for counting the particles contained in the plurality of droplets based on the light emission received by the light receiving means.
Two or more of the light receiving means are provided, and each of the light receiving means receives light emitted from the particles from different directions.
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.
The particle counting means counts the particles contained in the droplet based on the brightness value of the emission, and the particle counts the droplet based on the shape information on the light receiving surface of the emission. Perform at least one of the second counting processes for counting the contained particles,
A particle counting device, characterized in that, when the particle counting means performs the first counting process and determines that the particles cannot be counted, the particle counting means performs the second counting process and counts the particles. A light irradiation means that irradiates a plurality of droplets containing particles that can emit light when irradiated with light and that are ejected from different positions with the light.
A light receiving means that receives light emitted from the particles irradiated with the light, and a light receiving means.
A particle counting means for counting the particles contained in the plurality of droplets based on the light emission received by the light receiving means, and a particle counting means.
Have,
Two or more of the light receiving means are provided, and each of the light receiving means receives light emitted from the particles from different directions.
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.
The particle counting means counts the particles contained in the droplet based on the brightness value of the emission, and the particle counts the droplet based on the shape information on the light receiving surface of the emission. Perform at least one of the second counting processes for counting the contained particles,
The particle counting means calculates the radius of curvature based on the shape information of the light emitting surface on the light receiving surface, and counts the number of centers of the circles within a predetermined range as the number of particles. A particle counting device, characterized in that the second counting process is performed. One of claims 21 to 22, wherein 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. The particle counting device according to the description. The particle counting device according to any one of claims 21 to 23, 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 21 to 24, wherein the light emitted from the light irradiating means is pulsed light. The particle counting device according to any one of claims 21 to 25, wherein the particles capable of emitting light when irradiated with the light are cells. The particle counting device according to any one of claims 21 to 26, wherein the particles capable of emitting light when irradiated with light are at least one of a cell stained with a fluorescent dye and a cell capable of expressing a fluorescent protein. The particle counting device according to any one of claims 21 to 27,
A droplet forming apparatus comprising: a droplet ejection means having a plurality of ejection ports as an ejection start point. The droplet forming apparatus according to claim 28, wherein the plurality of ejection ports are arranged in one or more rows along the length direction of the ejection surface of the droplet ejection means. The droplet forming apparatus according to claim 29, wherein the number of the plurality of ejection ports is 2 or more and 100 or less per row. The droplet ejection means
A liquid holding portion that holds a liquid containing particles that can emit light when irradiated with the light, and a liquid holding portion.
6. Droplet forming device. The droplet forming apparatus according to claim 31, wherein the liquid holding portion has an open portion to the atmosphere. A light irradiation step of irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejecting from different positions with the light.
A light receiving step of receiving light emitted from the particles irradiated with the light, and a light receiving step.
A particle counting step of counting the particles contained in the plurality of droplets based on the light emitted received in the light receiving step is included.
The light receiving step is performed by two or more light receiving means, and each of the light receiving means receives light emitted from the particles from different directions.
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.
In the particle counting step, the first counting process for counting the particles contained in the droplet based on the brightness value of the emission, and the shape information on the light receiving surface of the emission are used for the droplet. Perform at least one of the second counting processes for counting the contained particles,
A particle counting method characterized in that, in the particle counting step, the first counting process is performed, and when the particles cannot be counted, the second counting process is performed to count the particles. A light irradiation step of irradiating a plurality of droplets containing particles capable of emitting light when irradiated with light and ejecting from different positions with the light.
A light receiving step of receiving light emitted from the particles irradiated with the light, and a light receiving step.
A particle counting step of counting the particles contained in the plurality of droplets based on the light emitted received in the light receiving step, and a particle counting step of counting the particles contained in the plurality of droplets.
Including
The light receiving step is performed by two or more light receiving means, and each of the light receiving means receives light emitted from the particles from different directions.
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.
In the particle counting step, the first counting process for counting the particles contained in the droplet based on the brightness value of the emission, and the shape information on the light receiving surface of the emission are used for the droplet. Perform at least one of the second counting processes for counting the contained particles,
In the particle counting step, the radius of curvature is calculated based on the shape information of the light emitting surface on the light receiving surface, and the number of centers of the circle when the radius of curvature is calculated within a predetermined range is counted as the number of particles. A particle counting method comprising performing the second counting process. The droplet forming apparatus according to any one of claims 28 to 32,
A dispensing device comprising: an adhered object having a plurality of recesses on which the droplets ejected from the droplet ejection means are deposited. The dispensing device according to claim 35, which has a control means for controlling the relative positional relationship between the droplet ejection means and the plurality of recesses. When the particle counting device in the droplet forming device determines that the number of particles contained in the droplet is 0,
The dispensing device according to any one of claims 35 to 36, wherein the droplet discharging means discharges the droplet again into the same recess. The dispensing device according to any one of claims 35 to 37, which is used for forming an organization.

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