JP2018017700A - Particle counter and particle counting method, droplet formation device, and dispensation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle counter that improves detection accuracy of particles contained in discharged droplets and has high productivity capable of detecting the number of particles in the droplets even when the number of droplets discharged per unit time is increased.SOLUTION: A particle counter 1 includes: light irradiation means 30 for irradiating with light L a plurality of droplets 210 that contain particles 200 capable of emitting light when irradiated with the light L and are discharged from different places; light receiving means 41 for receiving emitted light Lf from the particles 200 irradiated with the light; and particle number counting means 50 for counting the particles 200 contained in the plurality of droplets 210 based on the emitted light Lf received by the light receiving means 41.SELECTED DRAWING: Figure 1

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 advancement of stem cell technology, development of technology for forming a tissue body by ejecting a plurality of cells by inkjet has been performed. When discharging a droplet containing particles typified by cells, it is important to detect how many particles are included in the discharged droplet.

  As an apparatus having such a function, for example, it has a discharge head that pressurizes a diaphragm of a liquid chamber by a piezoelectric element and discharges a liquid containing cells from a nozzle, and irradiates the discharged liquid droplet with laser light. In addition, there has been proposed an ejection device that measures the number of cells in the ejected droplets by photographing or measuring the amount of light with a CCD camera or a photodiode (see, for example, Patent Document 1).

  The present invention improves the detection accuracy of the particles contained in the discharged droplets and has high productivity capable of detecting the number of particles in the droplets even when the number of droplets discharged per unit time is increased. An object is to provide a particle counter.

The particle counting apparatus of the present invention as means for solving the above-described problems includes particles that can emit light when irradiated with light, and irradiates light onto a plurality of droplets ejected from different positions. Light irradiation means;
A light receiving means for receiving light emitted from the particles irradiated with light;
Particle counting means for counting particles contained in the plurality of droplets based on light emission received by the light receiving means.

  According to the present invention, the detection accuracy of particles contained in discharged droplets is improved, and the productivity is high so that the number of particles in a droplet can be detected even when the number of discharged droplets per unit time is increased. A particle counting device can be provided.

FIG. 1 is a schematic view showing an example of a 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 openings 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 a plurality of ejection openings 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 a plurality of ejection openings 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 a plurality of ejection openings 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 a plurality of ejection openings 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 two light receiving means in the droplet forming apparatus shown in FIG. FIG. 13 is an explanatory diagram of light emission images obtained by the two light receiving means having the positional relationship shown in FIG. FIG. 14A is an explanatory diagram illustrating an example of a simulation image of a luminance value of light emission and a shape of a light-receiving surface when two light emissions overlap. FIG. 14B is an explanatory diagram illustrating another example of the simulation value of the luminance value of light emission and the shape of the light-receiving surface when the two light emissions overlap. FIG. 14C is an explanatory diagram illustrating another example of the simulation value of the luminance value of light emission and the shape of the light-receiving surface when the two light emissions overlap. FIG. 15A is an explanatory diagram illustrating an example of a simulation image of a luminance value of light emission and a shape on a light receiving surface of light emission when particles exist in the vicinity of the outer edge of the droplet. FIG. 15B is an explanatory diagram showing another example of the luminance value of light emission and the simulation image of the shape on the light receiving surface when the particle is present near the outer edge of the droplet. FIG. 16A is an image showing an example of the luminance value of light emission received by the light receiving means and the shape of the light emission on the light receiving surface. FIG. 16B is an image showing another example of the luminance value of light emission received by the light receiving means and the shape of the light receiving surface of the light emission. FIG. 16C is an image showing another example of the luminance value of light emission received by the light receiving means and the shape of the light receiving surface of the light emission. FIG. 17 is a graph showing an example of a calibration curve for determining the number of particles based on the luminance value of light emission received by two or more light receiving means. FIG. 18 is a flowchart showing an example of a flow for counting particles contained in 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 for counting particles contained in 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 for counting particles contained in 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 for counting particles contained in 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 for counting particles contained in 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 a flow for counting particles contained in 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 a dispensing apparatus of the present invention. FIG. 25 is a flowchart showing an example of the operation of the dispensing apparatus of the present invention. FIG. 26 is a flowchart showing another example of the operation of the dispensing apparatus of the present invention.

(Particle counting device and particle counting method)
The particle counting device of the present invention includes a light irradiation unit, a light receiving unit, and a particle counting unit, and further includes other units as necessary.

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

The particle counting method of the present invention can be preferably implemented by the particle counting apparatus of the present invention, the light irradiation step can be performed by a light irradiation unit, the light receiving step can be performed by a light receiving unit, 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 according to the present invention has a low productivity due to the small number of droplets discharged per unit time in the discharge device described in Patent Document 1 having a conventional single nozzle (one discharge port). Discharge droplets containing cells having a short lifetime as a disadvantage in forming a cell tissue within a limited time in which the cells are alive, and in the discharge device described in Patent Document 1, The 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. Therefore, the detection accuracy is low and false detection may occur. It is based on the knowledge that.

<Light irradiation means and light irradiation process>
The light irradiation means is means for irradiating light to a plurality of liquid droplets that contain particles that can emit light when irradiated with light and that are ejected from different positions.
The light irradiation step is a step of irradiating light to a plurality of droplets containing particles that can emit light when irradiated with light and discharged from different positions, and can be suitably performed by a light irradiation unit. it can.

The different positions mean that the discharge start points of a plurality of discharged droplets are different, that is, there are a plurality of droplet discharge start points. When the particle counting device is a droplet forming device having droplet discharge means, the plurality of discharge start points correspond to a plurality of discharge ports described later.
The plurality of discharge start points are preferably arranged in one or more rows along the direction orthogonal to the discharge direction of the plurality of droplets, and more preferably in the range of 1 to 4 rows.
The number of discharge start points 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 More than the number is more preferable.

There is no restriction | limiting in particular as a light irradiation means, According to the objective, it can select suitably, For example, a solid laser, a semiconductor laser, a pigment | dye laser, etc. are mentioned.
Examples of the solid laser include a YAG laser, a ruby laser, and a glass laser.
Examples of commercially available YAG lasers include Explorer ONE-532-200-KE (Spectra Physics Co., Ltd.).
There is no restriction | limiting in particular as a spot diameter of a laser, Although it can select suitably according to the objective, 100 micrometers or more and 2,000 micrometers or less are preferable. When the spot diameter is 100 μm or more and 2,000 μm or less, the probability of laser irradiation to the droplets increases even when there are variations in the ejection of flying droplets, so the counting accuracy of particles in multiple droplets decreases. There is an advantage that can be suppressed.

The light emitted from the light irradiation means is preferably pulsed light. Thereby, the counting accuracy of the number of particles in the droplet can be improved.
There is no restriction | limiting in particular as pulse width of pulsed light, Although it can select suitably according to the objective, 10 microseconds or less are preferable and 1 microsecond or less are more preferable.
The energy per unit pulse is not particularly limited and can be appropriately selected depending on the purpose. The energy per unit pulse is largely dependent on the optical system such as the presence or absence of light collection, but is preferably 0.1 μJ or more, more preferably 1 μJ or more. .

The light irradiation means irradiates a plurality of droplets in flight with light. Note that “in flight” refers to a state from when a droplet is ejected until the droplet is deposited on an object to be deposited.
As the light irradiation means, it is preferable that light can be irradiated in synchronization with the discharge of a plurality of droplets. Thereby, light can be more reliably irradiated to a plurality of droplets ejected from different positions.
Here, “synchronized” means that the light irradiating means irradiates light when a droplet is discharged and reaches a predetermined position. That is, the light irradiation means irradiates light with a delay of a predetermined time with respect to the discharge of the droplet.
It is preferable that the light irradiated from the light irradiation means is applied to one droplet in flight. When multiple droplets are ejected simultaneously from different positions and light is irradiated to multiple droplets at the same time, light emission from particles that can emit light when irradiated with light contained in multiple droplets is received simultaneously 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 a droplet, it is preferable to shift the timing at which a plurality of droplets are ejected from different positions.
In addition, even when multiple droplets in flight are irradiated simultaneously, 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 simultaneously. can do.

  The particles that can emit light when irradiated with light are not particularly limited and can be appropriately selected according to the purpose. However, cells are preferable, and the number of particles in flying droplets is counted by receiving autofluorescence. From the standpoint, cells expressing fluorescent proteins, stained cells stained with fluorescent dyes, inorganic fine particles stained with fluorescent dyes, and organic polymer particles stained with fluorescent dyes are preferred. Cells expressing fluorescent proteins, fluorescent dyes Stained cells stained with are particularly preferred.

There is no restriction | limiting in particular as fluorescent protein, According to the objective, it can select suitably, For example, green fluorescent protein (GFP; Green Fluorescent Protein), red fluorescent protein (RFP; Red Fluorescent Protein), yellow fluorescent protein (YFP) ; Yellow Fluorescent Protein).
The fluorescent dye in the stained cells is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include cell tracker orange and cell tracker red.
There is no restriction | limiting in particular as an organic polymer particle dye | stained with the fluorescent pigment | dye, According to the objective, it can select suitably, For example, SPHERO Fluorescent Nile Red particles (The Bay Biosciences make, 1% (w / v) And a diameter of 10 μm to 14 μm).
The number of particles contained in the droplet is preferably 1 or more, and more preferably 1 or more and 5 or less. If cells as particles are not included in the droplet, the tissue at that portion is lost. If cells as particles are excessively contained in the droplet, oxygen and nutrients may be deficient, and the cell fixing rate may be reduced.

In addition, when the particles aggregate, by adjusting the concentration of the liquid particles containing the particles, the concentration of the particles in the liquid and the number of particles in the liquid follow the Poisson distribution. Can be adjusted as appropriate.
There is no restriction | limiting in particular as a liquid, According to the objective, it can select suitably, For example, ion-exchange water, distilled water, a pure water, physiological saline etc. are mentioned.

There is no restriction | limiting in particular as a diameter of a droplet, Although it can select suitably according to the objective, 25 micrometers or more and 150 micrometers or less are preferable. When the diameter of the droplet is 25 μm or more, the diameter of the particles to be included becomes appropriate, and the number of applicable particles increases. Further, when the diameter of the droplet is 150 μm or less, the ejection of the droplet becomes stable.
Moreover, it is preferable that R> 3r, where R is the droplet diameter and r is the particle diameter. When R> 3r, the relationship between the diameter of the particle and the diameter of the droplet is appropriate, and is not affected by the edge of the droplet, so that the particle counting accuracy is improved.
The liquid amount of the droplet is not particularly limited and can be appropriately selected according to the purpose, but is preferably 1,000 pL or less, and more preferably 100 pL or less.
The liquid amount of the liquid droplet can be measured by, for example, a method of calculating the liquid amount by obtaining the size of the liquid droplet from the image of the liquid droplet.

<Light receiving means and light receiving process>
The light receiving means is 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 suitably performed by a light receiving means.

  There is no restriction | limiting in particular as a light-receiving means, According to the objective, it can select suitably, For example, the camera etc. which have a one-dimensional element and a two-dimensional element are mentioned. Among these, a camera having a two-dimensional element is preferable. If the light receiving means is a camera having a two-dimensional element, it is advantageous in that it is easy to obtain not only the luminance value of light emission but also the shape on the light receiving surface of light emission.

Examples of the one-dimensional element include a photodiode and a photosensor. Among these, a photomultiplier tube and an avalanche photodiode are preferable. When 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) imaging element, and a gate CCD.
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 a high-sensitivity camera (pco.edge, sCMOS, manufactured by Tokyo Instruments Co., Ltd.).

  The light receiving means receives the fluorescence emitted from the particles by absorbing the light as excitation light when the particles capable of emitting light are contained when the plurality of droplets in flight are irradiated with light. Since the fluorescence is emitted from the particles in all directions, the light receiving means can be arranged at any position where 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 does not directly enter.

  It is preferable that two or more light receiving means are provided, and each light receiving means receives light emitted from the particles from different directions. By having two or more light receiving means, even if a light receiving state is received by one light receiving means, the other light receiving means can receive a state where the light emission does not overlap. Based on the light emission received by the light receiving means, the particles contained in the droplets can be counted with high accuracy.

Since light emission is emitted in all directions from particles that can emit light when irradiated with light, two or more light receiving means receive light emitted in different directions from particles that can emit light when irradiated with light. It can be placed at any possible position. Note that three or more light receiving means may be arranged at a position where light emitted in different directions from particles capable of emitting light when irradiated with light can be received. Each light receiving means may have the same specification or different specifications.
When there is one light receiving means, particles that can emit light when irradiated with a plurality of light are included in the flying droplet, the particles that can emit light when irradiated with light overlap. As a result, there is a possibility that the particle counting means may erroneously detect the number of particles that can emit light when the light contained in the droplet is irradiated (count error occurs), but two or more light receiving means are provided. Thus, it is possible to reduce the influence of overlapping light-emitting particles when irradiated with light.
The particle counting means described later can be executed by comparing the luminance value or area value of particles that can emit 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 count errors by adopting data indicating the maximum value among the luminance values or area values obtained from the respective light receiving means (particles). If the overlap occurs, both the luminance value and the area value are reduced). 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 a plurality of obtained shape data.

  Light emission from the particle irradiated with light is emitted from the particle in all directions. For this reason, the two or more light receiving means are not particularly limited as long as they can be arranged at positions where light emission can be received, and can be appropriately selected according to the purpose. It is preferable to be arranged at a position where the angle does not become 0 °. It is advantageous in that information in a state where there is little overlap of light emission can be obtained.

  It is preferable that at least one light receiving unit among the two or more light receiving units is arranged so that the light receiving direction thereof is positioned substantially perpendicular to the light receiving direction of the other light receiving units. As a result, when one light receiving means and another light receiving means are used, when selecting one of the information received by one light receiving means and the other light receiving means, there is little overlap of light emission. Information can be selected. In addition, substantially orthogonal means 80 degrees or more and 100 degrees or less.

  The light receiving direction in the light receiving means other than the above two light receiving means is not particularly limited and can be appropriately selected according to the purpose. When there are two or more light receiving means, when two or more light receiving means are arranged on the same plane, the angle formed by the light receiving directions of adjacent light receiving means is 360 ° by the number of light receiving means, etc. It is preferable that the angle is divided. For example, when three light receiving units are arranged on the same plane, it is preferable that the light receiving directions of adjacent light receiving units be 120 °.

The light receiving means is preferably arranged so that the light receiving direction is positioned substantially perpendicular to the droplet discharge direction, and all the light receiving means are positioned substantially orthogonal to the droplet discharge direction. It is more preferable that they are arranged as described above. This makes it easy to adjust the position of the light receiving means, which is advantageous in that the structure of the particle counter 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 does not directly enter.

  As the light receiving means, it is preferable to obtain the luminance value of light emission received in the substantially normal direction of the light receiving surface and the shape information on the light receiving surface of the light emission. Thereby, the particle counting means counts the particles contained in the droplet based on the first counting process for counting the particles contained in the droplet based on the luminance value of the light emission, and 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 particle counting accuracy can be improved.

The light receiving means is preferably capable of receiving light emission in synchronization with the discharge of a plurality of droplets. As a result, a plurality of droplets ejected from different positions are irradiated with light from the light irradiation means, and light emitted from the particles can be received more reliably.
Here, the term “synchronized” means, for example, the timing at which a droplet is irradiated with light when a plurality of droplets are ejected and reach a predetermined position, and the light-emitting particles emit light when irradiated with the light. Means that the light receiving means receives light emission. That is, the light receiving unit detects light emission with a predetermined time delay with respect to the ejection of a plurality of droplets from different positions and the light irradiation by the light irradiation unit.

If the light emitted from the particles is weaker than the light emitted by the light irradiation means, a filter that attenuates the wavelength region of the light may be provided 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 range including the wavelength of light.

  As described above, the light emitted from the light irradiation means is preferably pulsed light, but may be light that is continuously oscillated. In this case, it is preferable to perform control so that the light receiving unit can receive light emission at the timing when the continuously oscillated light is applied to the ejected flying droplet.

<Particle counting means and particle counting step>
The particle counting means is a means for counting particles contained in a plurality of droplets based on light emission received by the light receiving means.
The particle counting step is a step of counting particles contained in a plurality of droplets based on light emission received by the light receiving unit, and can be suitably performed by the particle counting unit.

The particle counting means counts particles contained in the droplet based on the first counting process for counting particles contained in the droplet based on the luminance value of light emission, and information on the shape of the light receiving surface of 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 unit will be described.

[First counting process for counting particles based on luminance value of light emission]
The first counting process for counting particles based on the luminance value of light emission is not particularly limited and can be appropriately selected according to the purpose. For example, (1) the light emission received by at least two light receiving means 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 largest number of emitted light among the number of emitted light received by the light receiving means as the number of particles. It is done.

  In the processing of (1) above, if the particles can transmit light, the number of particles can be determined using a calibration curve based on the total luminance value of the light even if the light is overlapped. In order to obtain emission brightness values from two or more light-receiving means, even if the particles do not transmit light emission, the particles are counted based on the emission brightness values where the emission does not overlap or the emission overlap is small. Can do.

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

Among these, the treatment (1) is preferable. It is advantageous in that the accuracy of counting the number of particles is increased when the first counting process is the process (1).
When performing the first counting process, the light receiving means may be a one-dimensional element or a two-dimensional element.

[Second Counting Process for Counting Particles Based on Shape Information on Light Receiving Surface]
There is no restriction | limiting in particular as 2nd counting processing which counts particles based on the information of the shape in the light-receiving surface of light emission, For example, (3) Shape in the light-receiving surface of light emission can be selected suitably. (4) a process for counting the number of centers of curvature radii within a predetermined range of the calculated curvature radii as the number of particles, and (4) the length of the outer periphery of the shape on the light receiving surface is predetermined. Processing for counting the number of light emission within the range, (5) processing for counting the number of inflection points on the outer periphery of the shape on the light receiving surface of the light emission, and (6) processing on the outer periphery of the shape on the light receiving surface of the light emission. For example, a process of counting the number of changes in the sign of the tangential slope as the number of particles.

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

  In the process (4), the radius of curvature in the process (3) is replaced with the length of the outer periphery of the shape of the light receiving surface for light emission.

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

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

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

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

[Order of first counting process and second counting process]
The order of the first counting process and the second counting process is not particularly limited and can be appropriately selected according to the purpose. However, the particle counting means cannot perform the first counting process and count the particles. When it is determined, it is preferable to perform the second counting process and count the particles.
Since the first counting process is faster than the second counting process, the first counting process is performed first. In the first counting process, it may be difficult to count particles with a calibration curve based on the luminance value of light emission, for example, due to a phenomenon in which light emission from the particle is reflected inside the spherical surface of the droplet. In this case, by providing a non-determinable region in the calibration curve in advance, it is determined that particles cannot be counted in the first counting process, and switching to the second counting process makes it possible to count the particles contained in the droplets. Can be improved.

When the particle counting means determines that the number of particles is zero, it is preferable to further discharge droplets. Thereby, a predetermined number of particles can be adhered to the adherend.
In addition, it is possible to prevent a droplet having zero particles from adhering to an adherend. Thereby, the contamination of the adherend can be prevented.

Further, when the particle counting means determines that the number of particles is one or more, the droplet counting unit determines that the number of particles is determined to be one or more and then adheres to the adherend. You may make it transfer to next processes, such as a process of discharging a droplet, in the position different from the made position.
Further, after ejecting a predetermined number of droplets from different positions, the luminance value of light emission recorded in the recording means described later and the shape information on the light-receiving surface of the light emission are read, and the particles contained in each ejected droplet May be counted.

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

<Other means and other processes>
Other means are not particularly limited and may be appropriately selected depending on the intended purpose. However, it is preferable to have an optical system and a recording means. Other steps are preferably performed by other means.

  The optical system is not particularly limited and can be appropriately selected according to the purpose. For example, a lens for condensing light emitted from the light irradiation means into a droplet, a light receiving means for filtering light, Examples thereof include a filter for facilitating light reception.

  The recording unit is not particularly limited as long as it can record the luminance value of the light emission received by the light receiving unit and the shape information on the light receiving surface of the light emission, and can be appropriately selected according to the purpose. For example, RAM (Random Access Memory) ) And the like.

(Droplet forming device)
The droplet forming apparatus of the present invention includes the particle counter of the present invention and a droplet discharge means, and further includes other means as necessary.

<Droplet ejection means and droplet ejection process>
The droplet discharge means is a means having a plurality of discharge ports as discharge start points.
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 counter of the present invention.

  The operation method of the droplet discharge means is not particularly limited and can be appropriately selected depending on the purpose. For example, the piezoelectric pressurization method using a piezoelectric element, the thermal method using a heater, or the liquid by electrostatic attraction. Inkjet heads using an electrostatic method or the like that pulls the ink. Among these, the piezoelectric pressurization method is preferable from the viewpoint of relatively small heat and electric field damage to the particles.

The droplet discharge means preferably includes a liquid holding portion, a film-like member, and a vibration member, and more preferably includes other members as necessary.
The droplet discharge means may be either an open head or a closed head.

-Liquid holding part-
The liquid holding unit is a unit that holds a liquid containing particles that can emit light when irradiated with light.
When the droplet discharge means is an open head, it has an air release part on the upper side. Note that the position of the atmosphere opening part is not limited to the upper part. The bubbles mixed in the liquid are configured to be discharged from the atmosphere opening portion.

There is no restriction | limiting in particular about the shape of a liquid holding | maintenance part, a magnitude | size, a material, and a structure, According to the objective, it can select suitably.
Examples of the material of the liquid holding part include stainless steel, nickel, aluminum, silicon dioxide, alumina, zirconia, and the like.
Among these, when cells or proteins are used as particles, it is preferable to use materials having low adhesion to cells and proteins.
Cell adhesion is generally said to be dependent on the contact angle of the material with water. When the material is highly hydrophilic or highly hydrophobic, the cell adhesion is low. Various metal materials and ceramics (metal oxide) can be used as the highly hydrophilic material, and fluorine resin or the like can be used as the highly hydrophobic material.
In addition to these, it is also conceivable to reduce cell adhesion by coating the material surface. For example, the surface of the material can be coated with the above-mentioned metal or metal oxide material, or can be coated with a synthetic phospholipid polymer that imitates a cell membrane (for example, Lipidure manufactured by NOF Corporation).

-Membrane 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 unit is discharged as droplets from the plurality of discharge ports by vibration due to the amplitude motion.
The film-like member is fixed to the lower end portion of the liquid holding portion when the droplet discharge means is an open head.
The film-like member is fixed to the upper end portion of the liquid holding portion when the droplet discharge means is a closed head.
The liquid held in the liquid holding portion is discharged as droplets from a plurality of discharge ports that are through holes by the vibration of the film member.

There is no restriction | limiting in particular about the planar shape of a membranous member, a magnitude | size, a material, and a structure, According to the objective, it can select suitably.
Examples of the planar shape of the film member include a circle, an ellipse, a rectangle, a square, and a rhombus.
As the material of the film-like member, it is preferable to use a material having a certain degree of hardness because the film-like member vibrates easily if it is too soft, and it is difficult to suppress vibration immediately when not discharging. Examples thereof include metals, ceramics, and polymer materials. Specific examples include stainless steel, nickel, aluminum, silicon dioxide, alumina, and zirconia. Among these, similarly to the liquid holding unit, when cells or proteins are used as particles, it is preferable to use a material having low adhesion to cells or proteins.

-Multiple outlets-
There are no particular restrictions on the number, arrangement, spacing (pitch), opening shape, opening size, etc. of the plurality of discharge ports, and they can be appropriately selected according to the purpose.
The number of discharge ports arranged is not particularly limited and may be appropriately selected according to the purpose. However, it is preferable that one or more rows are arranged along the length direction of the discharge surface of the droplet discharge means. 1 column or more and 4 columns or less are more preferable. By providing one or more rows of ejection ports, the number of droplets ejected per unit time can be increased, and at the same time, the rows can be changed according to the type of particles (for example, the type of cells). it can.
The number of discharge ports per row is not particularly limited and is appropriately selected depending on 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 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 particle counter having high productivity that can increase 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 purpose. The arrangement may be a regular arrangement (for example, a staggered arrangement) or an irregular arrangement. .
In the case where the plurality of ejection openings are in a plurality of rows, a partition member is provided between each row in order to prevent interference between droplets ejected from adjacent ejection ports and improve particle detection sensitivity. Is preferred. Examples of the partition member include a partition plate.
The plurality of discharge ports are preferably arranged 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 is appropriately selected according to the purpose. For example, it is preferably 50 μm or more and 1,000 μm or less.
There is no restriction | limiting in particular as opening shape of a some discharge outlet, According to the objective, it can select suitably, For example, circular, an ellipse, a square etc. are mentioned.
The average diameter of the plurality of discharge ports is not particularly limited and may be appropriately selected according to the purpose. However, in order to avoid particles from clogging the discharge ports, the average diameter may be twice or more than the particle size. preferable.
When the particle is, for example, an animal cell, particularly a human cell, the size of the human cell is generally not less than 5 μm and not more than 50 μm. 10 μm or more and 100 μm or less is preferable.
On the other hand, if the droplets are too large, it is difficult to achieve the purpose of forming microdroplets, and therefore the average diameter of the plurality of discharge 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 vibration member is a member that causes the film-like member to vibrate and discharges droplets from a plurality of discharge ports (nozzles).
The vibrating member is formed on the lower surface side of the film-like member when the droplet discharge means is an open head.
The vibrating member is formed on the upper surface side of the film-like member when the droplet discharge means is a closed head.
There is no restriction | limiting in particular about the shape of a vibration member, a magnitude | size, a material, and a structure, According to the objective, it can select suitably.
The shape of the vibration member is not particularly limited and can be appropriately designed according to the shape of the film member. For example, when the planar shape of the film member is a circle, in the case of a closed head, It is preferable to provide a circular vibration member. In the case of an open head, it is preferable to form a vibrating member having an annular shape (ring shape) around the plurality of ejection openings.

A piezoelectric element is preferably used as the vibration member. As the piezoelectric element, for example, a structure in which electrodes for applying a voltage to the upper surface and the lower surface of a piezoelectric material are provided. In this case, by applying a voltage between the upper and lower electrodes of the piezoelectric element from the driving means, a compressive stress is applied in the lateral direction of the film, and the film member can be vibrated in the vertical direction of the film.
There is no restriction | limiting in particular as a piezoelectric material, According to the objective, it can select suitably, For example, a lead zirconate titanate (PZT), a bismuth iron oxide, a niobate metal substance, barium titanate, or these materials And metals and different oxides added. Among these, lead zirconate titanate (PZT) is preferable.

<Other means>
As other means, for example, it is preferable to have a drive means.
The driving means is not particularly limited and may be appropriately selected depending on the purpose. For example, when the droplet discharge means is an inkjet head of a piezoelectric pressurization method, means for inputting a drive voltage to the droplet discharge means Etc. In this case, minute droplets can be ejected by the driving means deforming the piezoelectric element.

Here, an embodiment of a droplet forming apparatus of the present invention will be described in detail with reference to the drawings.
The droplet forming apparatus of the present invention corresponds to the particle counting apparatus of the present invention provided with the droplet discharge means, and is included in the droplet forming apparatus of the present invention. Therefore, the following embodiments of the droplet forming apparatus of the present invention are described below. Through the description, the embodiment of the particle counter of the present invention will also be described.
In addition, in each drawing, the same code | symbol is attached | subjected to the same component and the overlapping description may be abbreviate | omitted. In addition, the number, position, shape, and the like of the following constituent members are not limited to the present embodiment, and can be set to a preferable number, position, shape, and the like in carrying out the present invention.

<First Embodiment>
FIG. 1 is a schematic diagram illustrating an example of a droplet forming apparatus according to the first embodiment.
The droplet forming apparatus 1 shown in FIG. 1 includes a droplet discharge unit 10, a drive unit 20, a light irradiation unit 30, a light receiving unit 41, and a particle counting unit 50.
In FIG. 1, a state in which one droplet is discharged from one of the four discharge ports 14 is representatively shown. Four droplets 210 can be continuously discharged from the outlet 14.
In FIG. 1, only one light receiving means 41 is shown as a representative, but an arbitrary position where light emission from particles contained in four droplets discharged from the four discharge ports 14 can be received and Can be arranged in numbers.

The droplet discharge means 10 is a closed head in the present embodiment, and is formed with a liquid holding unit 11 and four discharge ports 14 and can emit light when irradiated with light held in the liquid holding unit 11. The liquid 201 containing 200 is provided with the film-like member 12 that discharges four droplets 210 from the four discharge ports 14 in the discharge direction indicated by D3 in FIG.
The liquid holding unit 11 can also be configured by dividing the liquid into four liquid chambers for each of the four discharge ports 14 by a partition.

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

The liquid holding unit 11 is a unit that holds the liquid 201 including the particles 200, and is formed of, for example, a metal such as stainless steel, nickel, or aluminum, silicon, ceramics, or the like.
Although four discharge ports 14 that are through holes are provided on the lower surface side of the liquid holding unit 11, the number of discharge ports is not limited to four. The four discharge ports 14 are arranged in a line and are configured to be able to irradiate the light L from the light irradiation means 30 to the four discharged droplets 210.

  The diameters of the four discharge ports 14 are preferably not less than twice the size of the particles 200, and more preferably not less than 10 μm and not more than 100 μm.

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 one 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 twelve discharge ports 14 are arranged in a line along the length direction of the discharge surface 11a. It is installed.
FIG. 9 is a schematic plan view showing an example of an arrangement state of the discharge ports 14 of the discharge surface 11a of the droplet discharge means 10, and a total of 36 discharge ports 14 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 ejection ports 14 on the ejection surface 11a of the droplet ejection means 10, and a total of 20 ejection ports 14 in three rows along the length direction of the ejection surface 11a. It is arranged (a so-called houndstooth arrangement).
FIG. 11 is a schematic plan view showing an example of the arrangement state of the discharge ports 14 of the discharge surface 11a of the droplet discharge means 10, and a total of 36 discharge ports 14 in three rows along the length direction of the discharge surface 11a. The partition member 25 is provided between each row.

  The film-like member 12 is a unit in which four discharge ports 14 are formed and the four liquid droplets 210 are discharged from the four discharge ports 14 by vibration of the liquid 201 including the particles 200 held in the liquid holding unit 11. In the present embodiment, since the droplet discharge means 10 is a closed head, it is fixed to the upper end portion of the liquid holding unit 11. The planar shape of the film-like member 12 is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include a circle, an ellipse, and a rectangle.

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

The light irradiation means 30 irradiates light to the four droplets 210 in flight. The light irradiation unit 30 can emit light in synchronization with the droplet discharge by the droplet discharge unit 10 (in synchronization with the drive signal supplied from the drive unit 20 to the droplet discharge unit 10). Note that “in flight” refers to a state from when the droplet 210 is ejected from the droplet ejection means 10 until the droplet is deposited on the deposition target.
Here, synchronizing means not emitting light at the same time when a droplet is ejected by the droplet ejecting means 10 (simultaneously with the driving means 20 supplying a driving signal to the droplet ejecting means 10), but the droplets fly. This means that the light irradiation means 30 emits light at the timing when the droplet is irradiated with light when it reaches a predetermined position. That is, the light irradiation unit 30 emits light with a predetermined time delay with respect to the discharge of the four droplets 210 by the droplet discharge unit 10 (the drive signal supplied from the drive unit 20 to the droplet discharge unit 10). .

  It is preferable that the light L emitted from the light irradiation means 30 is applied to one flying droplet. When four droplets 210 are simultaneously ejected from the four ejection ports 14 and the light L is simultaneously irradiated onto the four droplets 210, light emission from the particles 200 included in the four droplets is received simultaneously. For this reason, it is difficult to count the number of particles 200 included in each droplet. That is, in order to improve the accuracy of counting the number of particles in the droplets, 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 irradiation means 30 is preferably pulsed light. For example, a solid 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, and 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.

When the particles 200 are contained in four flying droplets, the light receiving means 41 receives fluorescence emitted by the particles 200 by absorbing light as excitation light. Since the fluorescence is emitted from the particle 200 in all directions, the light receiving means 41 can be arranged at any 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 irradiation means 30 does not directly enter.
As the light receiving means 41, for example, a one-dimensional element such as a photodiode or a photosensor can be used. However, 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 charge coupled device (CCD), a complementary metal oxide semiconductor (CMOS), or a gate CCD may be used.

  Since the light emitted from the particles 200 is weaker than the light emitted from the light irradiation means 30, a filter for attenuating the wavelength region of the light may be provided in front of the light receiving means 41 (on the light receiving surface side). Thereby, in the light receiving means 41, an image of the particles 200 can be obtained by light with very high contrast. As the filter, for example, a notch filter that attenuates a specific wavelength region including the wavelength of light can be used.

The particle counting unit 50 detects the number of particles 200 (including the case of zero) in the four droplets based on the information from the light receiving unit 41.
The particle counting means 50 may be configured to 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 a program recorded in the ROM or the like into the main memory and executing it by the CPU. However, part or all of the particle counting means 50 may be realized only by hardware. The particle counting means 50 may be physically configured by a plurality of devices.

For example, the particle counting unit 50 can detect the number of particles 200 by comparing the amount of light received by the light receiving unit 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 unit 41, the particle counting unit 50 performs a method of performing image processing for calculating the luminance value or area of the particle 200 based on the two-dimensional image obtained from the light receiving unit 41. It may be used. In this case, the particle counting means 50 calculates the luminance value or area value of the particles 200 by image processing, and compares the calculated luminance value or area value with a preset threshold value to thereby determine the number of particles 200. Can be detected. In addition, when using a two-dimensional element, non-ejection detection can be performed by taking an image of a droplet at the timing immediately before receiving light emission.

The particle 200 is preferably a cell, and more preferably at least one of a cell stained with a fluorescent dye and a cell capable of expressing a fluorescent protein. In these, the autofluorescence is received by the light receiving means 41, and the particle counting means 50 can detect the number of cells contained in the droplet.
Examples of fluorescent dyes include cell tracker orange and cell tracker red.
Examples of the fluorescent protein include green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and the like.

<Variation 1 of the first embodiment>
FIG. 2 is a schematic diagram illustrating an example of a droplet forming apparatus according to Modification 1 of the first embodiment. Note that in the first modification of the first embodiment, the same reference numerals are given to the same configurations as those in the already described embodiment, and the description thereof is 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 discharge nozzles are provided via the first lens 52 and the second lens 53. Even when droplets are simultaneously ejected from the outlet 14 and light is simultaneously irradiated onto 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 discharged from the four discharge ports 14. There is no particular limitation as long as it is a position that does not interfere with the dropping of the droplets or the droplets to be deposited. In FIG. 2, the position of the light receiving means 41 is an arbitrary position at which light emission from the particles 200 included in the four droplets 210 discharged from the four discharge ports 14 can be received, It can arrange | position to the position which does not become obstructive for the landing to a landing subject.

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 emission Lf1 from the particle 200 is redirected by the first lens 52, collected by the second lens 53, and received by the second lens 53. Thus, it is possible to form an image on one light receiving means constituting 41. Thereby, even if it is a case where light is simultaneously irradiated to four droplets, the number of particles 200 contained in each of the four droplets can be counted.
In addition, you may adjust by combining a mirror with a 1st and 2nd lens as needed.

As the first lens 42 and the second lens 43, it is preferable to use a lens having a high numerical aperture (NA). In particular, the objective lens and the fθ lens make light incident on the light receiving means 41 perpendicularly. This is preferable because it is less likely to depend on the location of the light receiving means 41. Here, the objective lens is a lens capable of obtaining an enlarged image with little 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 light incident angle to 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. Among 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 diagram illustrating an example of a droplet forming apparatus according to a second modification of the first embodiment. Note that in the second modification of the first embodiment, the same reference numerals are given to the same configurations as those in the embodiment described above, and the description thereof is omitted.

In the droplet forming apparatus 1B shown in FIG. 3, the difference from the droplet forming apparatus 1 of the first embodiment shown in FIG. 1 is that particles contained in four droplets 210 ejected from the four ejection ports 14 are used. Is emitted from different directions by the two light receiving means 41 and 42. Note that the number of discharge ports is not limited to four.
The droplet forming apparatus 1B includes a droplet discharge unit 10, a drive unit 20, a light irradiation unit 30, light receiving units 41 and 42, and a particle counting unit 50.
In FIG. 3, the positions and the number of the light receiving means 41 and 42 are merely examples, and arbitrary positions where light emission from the particles 200 included in the four droplets 210 discharged from the four discharge ports 14 can be received and Can be arranged in numbers.

  The droplet discharge means 10 is a closed head, and stores the liquid 201 including the particles 200 when the liquid holding unit 11 is irradiated with light. By the vibration of the vibrating member 13 disposed above the liquid holding unit 11 The four droplets 210 including the particles 200 can be discharged from the four discharge ports 14 in the discharge direction indicated by D3 in FIG.

  The driving unit 20 is electrically connected to the vibration member 13 of the droplet discharge unit 10, and applies a driving voltage to the vibration member 13 to cause the vibration member 13 to vibrate, thereby causing four liquids to be discharged from the four discharge ports 14. Drops 210 are ejected.

  The light irradiation unit 30 is electrically connected to the driving unit 20 and receives a synchronization signal from the driving unit 20. The light irradiation unit 30 to which the synchronization signal is input irradiates the four droplets 210 with the laser light L as light in accordance with the discharge timing of the four droplets 210 by the droplet discharge unit 10.

  The two light receiving means 41 and 42 are both electrically connected to the driving means 20 via the light irradiation 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 emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and information on the shape of the light receiving surface of the light emission Lf1 and the shape of the light receiving surface of the light emission Lf2.

  The particle counting means 50 is based on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 received by the two light receiving means 41 and 42, and information on the shape of the light emission surface of the light emission Lf1 and the shape of the light emission surface of the light emission Lf2. 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 discharge direction of the droplets 210 is the depth direction of the paper surface, and the adherend object 300 is disposed immediately below the discharge port 14. In FIG. 12, one of the four discharge ports is shown as a representative.
As shown in FIG. 12, the light irradiation means 30, the mirror 60, and the lens 70 are liquids in which the laser light L irradiated by the light irradiation means 30 is reflected by the mirror 60 and discharged in the discharge direction through the lens 70. It arrange | positions so that it may concentrate on a droplet (position of the discharge outlet 14 in FIG. 12).
The two light receiving means 41 and 42 are arranged on a plane whose normal is the ejection 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 light emission images obtained by the two light receiving means having the positional relationship shown in FIG. In FIG. 13, the luminance of light emitted by the light receiving means 41 and the shape of the light receiving surface of the light emission are shown in one image P1, and the luminance of light emitted by the light receiving means 42 and the shape of the light receiving surface of the light emission are shown in one image P2. Show.
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 luminance of light emission, either of the processes (1) or (2) may be performed to count the number of particles 200.
When the particles 200 are counted from the image P1 and the image P2 on the basis of the shape of the light-emitting light receiving surface, the number of particles 200 may be counted by performing any one of the processes (3) to (6).

  FIG. 14A to FIG. 14C are explanatory diagrams illustrating an example of a simulation image of a luminance value of light emission and a shape of a light-receiving surface when two light emissions overlap. In FIGS. 14A to 14C, particles that can transmit light are assumed, and the density of the image indicates the luminance of light emission, and the lighter the color, the higher the luminance of light emission.

As shown in FIG. 14A, the number of particles can be determined based on the luminance value of light emission as long as the light emission from the particles overlaps.
As shown in FIG. 14B, when the number of particles cannot be determined on the basis of the luminance value of light emission when the light emission from the particles overlaps, the curvature of the shape on the light receiving surface based on the shape on the light receiving surface. The radius is calculated, and the number of centers of the calculated curvature radius is determined as the number of particles.
As shown in FIG. 14C, even when light emission from particles completely overlaps, it can be determined that the number of overlapping particles is larger as the luminance is higher, so the number of particles can be determined using a calibration curve. .
In addition, even in the case of particles that do not transmit light, by having two or more light receiving means, if any light receiving means can receive a state in which light emission does not overlap, unless the particles are in contact with each other, The number of particles can be determined based on the luminance value of light emission.

FIG. 15A and FIG. 15B are explanatory diagrams illustrating an example of a simulation image of the luminance of light emission and the shape on the light receiving surface of the light emission when particles exist in the vicinity of the outer edge of the droplet. In FIGS. 15A and 15B, as in FIGS. 14A to 14C, the shade of the image indicates the luminance of light emission, and the lighter the color, the higher the luminance of light emission. However, it differs from FIGS. 14A to 14C in that one particle is included in the droplet and the particle is present near the outer edge of the droplet.
As shown in FIG. 15A, the outer edge of the droplet emits light, and when it is attempted to determine the number of particles based on the luminance value of light emission, it is determined whether the number of particles is one or two. Although difficult, if the number of particles is determined based on the shape of the light-receiving surface of the light emission, it can be excluded because the radius of curvature of light emission at the outer edge of the droplet is large.
As shown in FIG. 15B, as in the case of FIG. 15A, when it is attempted to determine the number of particles based on the luminance value of light emission, it is difficult to determine whether the number of particles is one or two. When the number of particles is determined on the basis of the shape of the light-receiving surface for light emission, it can be determined that the number is 1 from the radius of curvature of light emission.

  16A to 16C are images showing an example of the luminance value of light emission received by the light receiving means and the shape of the light receiving surface of the light emission. 16A to 16C, SPHERO Fluorescent Nile Red particles (manufactured by Bay Biosciences, 1% (w / v), diameter 10 μm to 14 μm) were used as particles, and ion exchange water was used as a liquid, and a piezoelectric element was used. The membrane vibration type inkjet head is driven with a sine wave voltage (20,000 Hz, 1 period, 4.6 V), and droplets containing particles are ejected into a sphere with a diameter of 80 μm, which is a high light receiving means. The exposure time of a sensitivity camera (pco.edge, manufactured by Tokyo Instruments Co., Ltd.) is set to 10 ms, and a YAG laser (Explorer ONE-532-200-KE, Spectra Physics Co., Ltd.) is used as a light irradiation means for the discharged droplets. Laser beam (10k) It is an image in a state where particles are emitted by irradiation with 100 μJ at Hz. The delay time was set to 0.1 ms so that the inkjet head, YAG laser, and high-sensitivity camera were synchronized and only the YAG laser was delayed. 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 properties of the liquid to be discharged, particles, and the like.

FIG. 16A shows that there are two particles in a spherical or ellipsoidal droplet with depth, one of the particles is in front of the paper in the droplet, and the other particle is in the droplet. It is an image when light is received from the light receiving direction that exists on the back side of the paper.
As shown in FIG. 16A, the light emission from the particles existing on the back side of the paper in the liquid droplet attenuates the luminance value of the light emission due to refraction or scattering by the amount that passes through the liquid droplet. There are times when the luminance value of the light emission varies due to the position in the drop.

FIG. 16B is an image when light is received from a light receiving direction in which three particles are present in a spherical or ellipsoidal droplet having a depth, and one of the particles is present near the outer edge of the droplet. is there.
As shown in FIG. 16B, light emission from particles present 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 spherical or ellipsoidal droplet having a depth, and the particle is received from the light receiving direction in the vicinity of the outer edge of the droplet on the back side of the paper. It is an image.
As shown in FIG. 16C, the light emission from the particles in the case where the particles are present in the vicinity of the outer edge of the droplet on the back side of the paper surface has a low luminance value, 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 light emission received by two or more light receiving means.
As shown in FIG. 17, by using a calibration curve for determining the number of particles based on the luminance value of emitted light, the luminance value 1 of emitted light received by the first light receiving unit and the light received by the second light receiving unit are received. Based on the luminance value 2 of the emitted light, the number of particles can be determined.
In addition, as shown in FIGS. 16A to 16C, the luminance value of light emission becomes unclear, and a 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 undecidable area. When plotted in a non-determinable area based on the brightness values 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.
Note that the calibration curve data is acquired in advance according to the type of particles, light emission conditions, and the like, and recorded in a recording means or the like.

Next, a flow for counting particles contained in a plurality of droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention is according to the step represented by S in the flowchart shown in FIG. This will be described with reference to FIG.
This flowchart is a procedure used when the light emission does not overlap or when the light emission does not become unclear due to reflection inside the spherical surface of the droplet.

  In S101, the droplet discharge unit 10 to which the drive voltage is applied from the drive unit 20 discharges the droplet 210 including the particles 200 from the discharge port (nozzle) 14 in the discharge direction indicated by D3 in FIG. The driving unit 20 applies a driving voltage to the droplet discharge unit 10 and outputs a synchronization signal to the light irradiation unit 30 and the two light receiving units 41 and 42. Thereafter, the particle counting unit 50 shifts the process to S102.

  In S <b> 102, the droplet discharge unit 10 to which the synchronization signal is input from the driving unit 20 irradiates the droplet 210 with laser light L as light in accordance with the discharge timing of the droplet 210. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2. Thereafter, the particle counting means 50 shifts the process to S103.

  In S103, the particle counting unit 50 receives the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2 received by the two light receiving units 41 and 42. Based on the above, the particle 200 contained in the droplet 210 is counted using the calibration curve shown in FIG.

Next, a flow for counting particles contained in a plurality of droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention is according to the step represented by S in the flowchart of FIG. This will be described with reference to FIG.
This flowchart is a procedure used when, for example, the number of particles 200 included in the droplets 210 ejected by the droplet ejection unit 10 varies for each ejection. That is, in order to confirm the degree of variation in the number of particles 200, the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 from the particle 200, the shape of the light emission Lf1 on the light receiving surface, and the shape of the light emission Lf2 on the light receiving surface each time it is ejected. Is obtained, and the number of particles 200 is counted based only on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2.

  In S201, the droplet discharge unit 10 to which the drive voltage is applied from the drive unit 20 discharges the droplet 210 including the particles 200 from the discharge port 14 in the discharge direction indicated by D3 in FIG. The driving unit 20 applies a driving voltage to the droplet discharge unit 10 and outputs a synchronization signal to the light irradiation unit 30 and the two light receiving units 41 and 42. Thereafter, the particle counting means 50 moves the process to S202.

  In S <b> 202, the droplet discharge unit 10 that has received the synchronization signal from the driving unit 20 irradiates the droplet 210 with laser light L as light in accordance with the discharge timing of the droplet 210. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2. Thereafter, the particle counting means 50 moves the process to S203.

  In S203, the particle counting unit 50 uses the calibration curve shown in FIG. 17 to determine the particles contained in the droplet 210 based on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 received by the two light receiving units 41 and 42. Count 200. Thereafter, the particle counting means 50 moves the process to S204.

  In S204, the particle counting unit 50 that has counted the particles 200 contained in the droplets 210 determines whether or not the droplet discharging unit 10 has ejected the droplets 210 a predetermined number of times. When the particle counting unit 50 determines that the droplets 210 have been ejected a predetermined number of times by the droplet ejecting unit 10, the process ends. If the particle counting means 50 determines that the droplet ejection means 10 has not ejected the droplet 210 a predetermined number of times, it returns the process to S201.

Next, a flow for counting particles contained in a plurality of droplets ejected from the droplet ejection means using the droplet forming apparatus of the present invention is a step represented by S in FIG. 20 in the flowchart shown in FIG. Therefore, description will be made with reference to FIG.
This flowchart is a procedure used when, for example, it is desired to check how many particles 200 are contained in the continuously discharged droplets 210 after the droplets 210 are continuously discharged by the droplet discharge means 10. is there.

  In S301, the droplet discharge unit 10 to which the drive voltage is applied from the drive unit 20 discharges the droplet 210 including the particles 200 from the discharge port 14 in the discharge direction indicated by D3 in FIG. The driving unit 20 applies a driving voltage to the droplet discharge unit 10 and outputs a synchronization signal to the light irradiation unit 30 and the two light receiving units 41 and 42. Thereafter, the particle counting means 50 moves the process to S302.

  In S <b> 302, the droplet discharge unit 10 to which the synchronization signal is input from the driving unit 20 irradiates the droplet 210 with laser light L as light in accordance with the discharge timing of the droplet 210. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2. The particle counting unit 50 records the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 and the information on the shape of the light receiving surface of the light emission Lf1 and the shape of the light receiving surface of the light emission Lf2 on a recording unit (not shown) for each droplet 210, The process proceeds to S303.

  In S303, the particle counting unit 50 that has counted the particles 200 contained in the droplets 210 determines whether or not the droplet discharging unit 10 has discharged the droplets 210 a predetermined number of times. If the particle counting unit 50 determines that the droplet 210 has been ejected a predetermined number of times by the droplet ejection unit 10, the process proceeds to S304. If the particle counting means 50 determines that the droplet ejection means 10 has not ejected the droplet 210 a predetermined number of times, it returns the process to S301.

  In S304, the particle counting means 50 determined to have ejected the droplet 210 a predetermined number of times, the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 recorded in the recording means, the shape of the light emission Lf1 on the light receiving surface and the light 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.

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

  In step S401, the droplet discharge unit 10 to which the drive voltage is applied from the drive unit 20 discharges the droplet 210 including the particles 200 from the discharge port 14 in the discharge direction indicated by D3 in FIG. The driving unit 20 applies a driving voltage to the droplet discharge unit 10 and outputs a synchronization signal to the light irradiation unit 30 and the two light receiving units 41 and 42. Thereafter, the particle counting means 50 moves the process to S402.

  In S <b> 402, the droplet discharge unit 10 to which the synchronization signal is input from the driving unit 20 irradiates the droplet 210 with the laser light L as light in accordance with the discharge timing of the droplet 210. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2. Thereafter, the particle counting means 50 moves the process to S403.

  In S403, the particle counting means 50 uses the calibration curve shown in FIG. 17 for each droplet 210 based on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 received by the two light receiving means 41 and 42. Determine the number of. At this time, if the particle counting unit 50 determines that the luminance value of the received light emission Lf2 is not included in the non-determinable area shown in FIG. 17, the process proceeds to S404. If the particle counting unit 50 determines that the particle is included in the non-determinable area, the process proceeds to S405.

  In S404, the particle counting means 50 determined not to be included in the non-determinable region is based on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 received by the two light receiving means 41 and 42, and the calibration curve shown in FIG. Is used to count the particles 200 contained in the droplet 210, and the process is terminated.

In S405, the particle counting unit 50 determined to be included in the non-determinable region receives information on the shape of the light receiving surface of the light emission Lf1 and the shape of the light receiving surface of the light emission Lf2 received by the two light receiving units 41 and 42, that is, shape data. Based on the above, the radius of curvature is obtained.
Next, the particle counting means 50 determines whether or not the calculated curvature radius is within a predetermined range. If the particle counting unit 50 determines that all the calculated radii of curvature are within the predetermined range, the process proceeds to S406, and if it is determined that even one of the calculated radii of curvature is not within the predetermined range, the process is performed. The process proceeds to S404.

  In S <b> 406, the particle counting unit 50 that determines that all the radii of curvature are within a predetermined range counts the number of the centers of the calculated radii of curvature as the number of particles 200 included in the droplet 210, and ends the present process. To do.

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

  In S501, the droplet discharge unit 10 to which the drive voltage is applied from the drive unit 20 discharges the droplet 210 including the particles 200 from the discharge port 14 in the discharge direction indicated by D3 in FIG. The driving unit 20 applies a driving voltage to the droplet discharge unit 10 and outputs a synchronization signal to the light irradiation unit 30 and the two light receiving units 41 and 42. Thereafter, the particle counting means 50 moves the process to S502.

  In S <b> 502, the droplet discharge unit 10 to which the synchronization signal is input from the drive unit 20 irradiates the droplet 210 with the laser beam L as light in synchronization with the discharge timing of the droplet 210. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2. Thereafter, the particle counting means 50 moves the process to S503.

  In S503, the particle counting means 50 uses the calibration curve shown in FIG. 17 based on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 received by the two light receiving means 41 and 42. Count 200. Thereafter, the particle counting means 50 moves the process to S504.

  In S504, the particle counting means 50 determines whether or not the number of counted particles 200 is zero. If the particle counting means 50 determines that the counted particles 200 are zero, the process proceeds to S501. If the particle counting means 50 determines that the counted particles 200 are not zero, the present processing is terminated.

  Next, using the droplet forming apparatus of the present invention, when the particle counting unit determines that the number of particles included in the plurality of discharged droplets is zero, the droplet discharging unit discharges the droplet again. The flow that is repeated until the particles 200 are counted will be described with reference to FIG. 3 and the like according to the step represented by S in the flowchart of FIG.

  In step S601, the droplet discharge unit 10 to which the drive voltage is applied from the drive unit 20 discharges the droplet 210 including the particles 200 from the discharge port 14 in the discharge direction indicated by D3 in FIG. The driving unit 20 applies a driving voltage to the droplet discharge unit 10 and outputs a synchronization signal to the light irradiation unit 30 and the two light receiving units 41 and 42. Thereafter, the particle counting unit 50 moves the process to S602.

  In S <b> 602, the droplet discharge unit 10 to which the synchronization signal is input from the driving unit 20 irradiates the droplet 210 with the laser beam L as light in synchronization with the discharge timing of the droplet 210. The light receiving means 41 and 42 to which the synchronization signal is input receive the light emission Lf1 and the light emission Lf2 in accordance with the timing at which the particles 200 emit light by the laser light L of the light irradiation means 30. Thereby, the light receiving means 41 and 42 obtain the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2, and the shape information on the light receiving surface of the light emission Lf1 and the shape on the light receiving surface of the light emission Lf2. Thereafter, the particle counting unit 50 moves the process to S603.

  In S603, the particle counting unit 50 uses the calibration curve shown in FIG. 17 based on the luminance value of the light emission Lf1 and the luminance value of the light emission Lf2 received by the two light receiving units 41 and 42. Count 200. Thereafter, the particle counting unit 50 moves the process to S604.

  In S604, the particle counting unit 50 that has counted the particles 200 determines whether or not the number of counted particles 200 is zero. If the particle counting means 50 determines that the counted particles 200 are zero, the process proceeds to S605. If the particle counting means 50 determines that the counted particles 200 are not zero, the present processing is terminated.

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

<Second Embodiment>
FIG. 4 is a schematic diagram illustrating an example of a droplet forming apparatus according to the second embodiment. Note that in the second embodiment, the same components as those described in the above embodiments are denoted by the same reference numerals, and the description thereof is 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 discharge means 10 is changed to an open head.
The droplet forming apparatus 1C shown in FIG. 4 includes a droplet discharge unit 10, a drive unit 20, a light irradiation unit 30, a light receiving unit 41, and a particle counting unit 50.
FIG. 4 representatively shows a state in which one droplet 210 is discharged from one of the three discharge ports 14. However, by appropriately adjusting the timing of discharging the droplet, It is possible to discharge continuously from the discharge port 14. Note that the number of discharge ports is not limited to three.
In FIG. 4, the position and number of the light receiving means 41 are merely examples, and the light receiving means 41 is arranged at any position and number where light emission from particles contained in the three droplets 210 discharged from the three discharge ports 14 can be received. can do.

The droplet discharge means 10 is an open head in this embodiment, and includes a liquid holding unit 11 and three discharge ports 14, and the liquid 201 held in the liquid holding unit 11 is separated into three by vibration of the vibration member 13. And a film-like member 5 that discharges as three droplets 210 from the discharge port 14.
The liquid holding unit 11 is a unit that holds the liquid 201 including the particles 200 that can emit light when irradiated with light. In the present embodiment, the liquid holding unit 11 is an open head, and thus has an air release unit 15 at the top. . By this, the air bubbles mixed in the liquid 201 can be discharged from the atmosphere opening portion.

In this embodiment, the vibrating member 13 is disposed on the lower surface of the film-like member 12 because the droplet discharge means 10 is an open head. The shape of the vibration 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 12 is circular, it is preferable to form the vibrating member 13 having an annular shape (ring shape) around the three discharge ports 14.
By supplying a driving signal from the driving means 20 to the vibrating member 13, the film-like member 12 can be vibrated. Thereby, three droplets 210 can be discharged from the three discharge ports 14.

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

The driving means 20 selectively causes the vibrating member 13 to generate a discharge waveform that forms a droplet by vibrating the film-like member 12 and a stirring waveform that vibrates the film-like member 12 within a range where no droplet is formed (for example, (Alternately).
That is, the driving unit 20 applies the discharge waveform to the vibrating member 13 and controls the vibration state of the film-like member 12, thereby causing the liquid 201 including the particles 200 held by the liquid holding unit 11 to flow from the three discharge ports 14. It can be discharged as three droplets. Further, the driving unit 20 can stir the liquid 201 including the particles 200 held by the liquid holding unit 11 by adding a stirring waveform to the vibrating member 13 and controlling the vibration state of the film-like member 12. Note that no droplets are discharged from the three discharge ports 14 during stirring.

  In this way, by stirring the liquid 201 including the particles 200 held in the liquid holding unit 11 while no droplets are formed, the particles 200 are prevented from settling and aggregating on the film-like member 12. The particles 200 can be uniformly dispersed in the liquid 201. Thereby, it is possible to suppress the clogging of the discharge port and the variation in the number of particles 200 in the discharged droplet. As a result, the liquid 201 containing the particles 200 can be stably discharged as droplets continuously for a long time.

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

<Modification Example 1 of Second Embodiment>
FIG. 5 is a schematic diagram illustrating an example of a droplet forming apparatus according to Modification 1 of the second embodiment. Note that in the first modification of the second embodiment, the same reference numerals are assigned to the same configurations as those in the embodiment described above, and the description thereof is omitted.

  A droplet forming apparatus 1D of the first modification of the second embodiment shown in FIG. 5 corresponds to the droplet forming apparatus 1A of the first modification of the first embodiment shown in FIG. Since it is the same as the droplet forming apparatus 1A of the first modification of the first embodiment except that it is changed to an open head, refer to the description of the second embodiment and the first modification of the first embodiment. Detailed description will be omitted.

<Modification 2 of the second embodiment>
FIG. 6 is a schematic diagram illustrating an example of a droplet forming apparatus according to Modification 2 of the second embodiment. Note that in the second modification of the second embodiment, the same reference numerals are given to the same configurations as those in the embodiment described above, and the description thereof is omitted.

  A droplet forming apparatus 1E of Modification 2 of the second embodiment shown in FIG. 6 corresponds to the droplet forming apparatus 1B of Modification 2 of the first embodiment shown in FIG. Except for the change to the open head, it is common to the droplet forming apparatus 1B of the second modification of the first embodiment, so refer to the description of the second embodiment and the second modification of the first embodiment. Detailed description will be omitted.

  The droplet forming apparatus of the present invention has high productivity that improves the detection accuracy of particles contained in the discharged droplets and can increase the number of droplets discharged per unit time. It is preferably used in various fields, but is particularly preferably used in the dispensing device of the present invention described below.

(Dispensing device)
The dispensing apparatus of the present invention includes the droplet forming apparatus of the present invention and an object to be deposited, preferably has 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 zero, the droplet discharging means may discharge the droplet again to the same recess. This is preferable because the particles can be surely dispensed.

<Object to be deposited>
The adherend is a member formed with a plurality of recesses where droplets ejected from the droplet ejecting means of the droplet forming apparatus are deposited.
The material to be deposited is not particularly limited as to the material, shape, size, structure, and the like as long as the ejected liquid droplets can adhere thereto, and can be appropriately selected according to the purpose.
The material of the adherend is not particularly limited and may be appropriately selected depending on the purpose. For example, a material formed of a semiconductor, ceramics, metal, glass, quartz glass, plastics, or the like is preferable. It is done.
There is no restriction | limiting in particular as a shape of a to-be-adhered object, Although it can select suitably according to the objective, For example, plate shape, plate shape, etc. are preferable.
There is no restriction | limiting in particular as a structure of a to-be-adhered object, According to the objective, it can select suitably, For example, a single layer structure or a multilayer structure may be sufficient.
The number of recesses provided in the adherend is plural, preferably 2 or more, more preferably 5 or more, and still more preferably 50 or more.

<Control means>
The control means is means for controlling the relative positional relationship between the droplet discharge 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. And perform various processes based on a control program for controlling the operation of the entire dispensing apparatus.

<Other means>
The other means is not particularly limited and may be appropriately selected depending on the intended purpose, but preferably has a recording means, a culture means, a heating means, a stirring means, a washing means, and the like.

  The dispensing device of the present invention improves the detection accuracy of particles contained in the ejected droplets, and increases the number of droplets ejected per unit time, and has high productivity. Since it has an apparatus, it can be suitably used for the production of tissue bodies, particularly three-dimensional tissue bodies, which can be widely used in various fields such as regenerative medicine, medicine, cosmetics, and evaluation of safety and efficacy of chemical substances.

  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 diagram illustrating an example of a dispensing apparatus according to the third embodiment. In the third embodiment, the droplet forming device of the first and second embodiments is used as a dispensing device that dispenses particles into the recesses of the adherend. Note that in the third embodiment, identical symbols are assigned to configurations identical to those of the previously described embodiments and descriptions thereof are omitted.

A dispensing apparatus 2 shown in FIG. 24 includes a droplet forming apparatus 1, a deposition target 300, a stage 400, and a control unit 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 already described in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.
Instead of the droplet forming apparatus 1, any one of the droplet forming apparatuses 1A to 1E shown in FIGS. 2 to 6 may be used.

The deposition target 300 is disposed on a stage 400 configured to be movable. A plurality of recesses (wells) 310 into which four droplets 210 ejected from the droplet ejection means 10 of the droplet forming apparatus 1 are deposited are formed on the deposition target 300.
The control unit 500 moves the stage 400 and controls the relative positional relationship between the droplet discharge unit 10 of the droplet forming apparatus 1 and the respective recesses 310. As a result, the droplets 210 including the particles 200 can be sequentially discharged into the respective recesses 310 from the droplet discharge means 10 of the droplet forming apparatus 1.

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

  FIG. 25 is an example of a flowchart showing the operation of the dispensing apparatus according to the third embodiment. First, in step S <b> 101, the droplet discharge means 10 of the droplet forming apparatus 1 discharges the droplet 210 toward the predetermined recess 310.

  Next, in step S <b> 102, the particle counting unit 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 unit 500. When 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 S <b> 103, the control unit 500 controls the stage 400 and moves the deposition target 300 so that the droplet discharge unit 10 of the droplet forming apparatus 1 and the next concave portion 310 are positioned to face each other. Then, it transfers to step S101 and repeats the same operation | movement.

  As a result, when the number of particles 200 contained in the droplets 210 flying toward the recesses 310 is zero, the droplets 210 are discharged again toward the same recesses 310. The particles 200 can be reliably dispensed.

Further, it is possible to detect the number of particles 200 contained in the flying droplet 210 as shown in FIG. 26, not 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 apparatus according to the third embodiment.

  In FIG. 26, first, after performing step S101 similar to FIG. 25, 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”.

  Note that if the number of particles 200 contained in the droplet 210 increases, the detection accuracy of the particle counting means 50 may decrease, so the number of particles 200 contained in the droplet 210 ejected at one time is not necessarily three. There is no need to set them individually. For example, the number of particles 200 contained in the droplets 210 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 becomes three.

  When 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. Then, it transfers to step S101 and repeats the same operation | movement. Thereby, it becomes possible to dispense so that the number of particles 200 in each recess 310 is three.

  In the processes 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 unit 500 as a program, for example.

As an aspect of this invention, it is as follows, for example.
<1> a light irradiation unit that includes particles capable of emitting light when irradiated with light, and irradiates the light to a plurality of droplets ejected from different positions;
A light receiving means for receiving light emitted from the particles irradiated with the light;
Particle counting means for counting the particles contained in the plurality of droplets based on the light emission received by the light receiving means;
It is a particle counter characterized by having.
<2> The particle counter according to <1>, wherein the particle counter includes two or more light receiving units, and each of the light receiving units receives light emitted from the particles from different directions.
<3> The light receiving unit obtains information on the luminance value of the light emission received in a substantially normal direction of the light receiving surface and the shape of the light emission on the light receiving surface,
A first counting process in which the particle counting means counts the particles contained in the droplet based on the luminance value of the light emission; and the droplet based on the shape information on the light receiving surface of the light emission. The particle counter according to <2>, wherein at least one of a second counting process for counting the particles contained therein is performed.
<4> The particle according to <3>, wherein when the particle counting unit performs the first counting process and determines that the particle cannot be counted, the second counting process is performed to count the particle. It is a counting device.
<5> The particle counting means calculates a radius of curvature based on information on the shape of the light emission on the light-receiving surface, and calculates the number of circle centers when the radius of curvature within a predetermined range is calculated. The particle counter according to any one of <3> to <4>, wherein the second counting process for counting the number is performed.
<6> From <2> to <2>, wherein at least one of the two or more light receiving means is disposed such that a light receiving direction thereof is positioned substantially perpendicular to a light receiving direction of the other light receiving means. 5>. The particle counter according to any one of 5>.
<7> The particle counter according to any one of <2> to <6>, wherein the light receiving unit is disposed such that a light receiving direction thereof is positioned in a direction substantially orthogonal to a droplet discharge direction.
<8> The particle counter according to any one of <1> to <7>, wherein the light irradiated from the light irradiation unit is pulsed light.
<9> The particle counter according to <8>, wherein the pulse width of the pulsed light is 10 μs or less.
<10> The particle counter according to any one of <1> to <9>, wherein the particles that can emit light when irradiated with light are cells.
<11> The particles according to any one of <1> to <10>, wherein the particles capable of emitting light when irradiated with light are at least one of cells stained with a fluorescent dye and cells capable of expressing a fluorescent protein. It is a particle counter of description.
<12> The particle counter according to any one of <1> to <11>,
Droplet discharge means having a plurality of discharge ports as discharge start points;
A droplet forming apparatus.
<13> The droplet forming apparatus according to <12>, wherein the plurality of discharge ports are disposed in one or more rows along a length direction of a discharge surface of the droplet discharge unit.
<14> The droplet forming apparatus according to <13>, wherein the number of the plurality of ejection openings is 2 or more and 100 or less per row.
<15> The droplet discharge means is
A liquid holding unit for holding a liquid containing particles capable of emitting light when irradiated with the light;
A plurality of discharge ports are formed, and a film-like member that discharges the liquid held in the liquid holding portion as droplets from the plurality of discharge ports by vibration,
The droplet forming apparatus according to any one of <12> to <14>.
<16> The liquid droplet forming apparatus according to <15>, wherein the liquid holding unit includes an atmosphere opening unit.
<17> The droplet forming apparatus according to any one of <15> to <16>, wherein the droplet discharge unit includes a vibrating member that vibrates the film-like member.
<18> The droplet forming device according to <17>, wherein the vibration member is a piezoelectric element.
<19> A light irradiation step that includes particles capable of emitting light when irradiated with light and irradiates the plurality of droplets ejected from different positions with the light,
A light receiving step for receiving light emitted from the particles irradiated with the light;
A particle counting step of counting the particles contained in the plurality of droplets based on the emitted light received in the light receiving step;
The particle counting method characterized by including.
<20> The droplet forming apparatus according to any one of <12> to <18>,
An object to be deposited on which a plurality of recesses to which the droplets ejected from the droplet ejection means are deposited; and
It is a dispensing device characterized by having.
<21> The dispensing device according to <20>, further including a control unit that controls a relative positional relationship between the droplet discharge unit and the plurality of recesses.
<22> When the particle counter in the droplet forming apparatus determines that the number of particles contained in the droplet is zero,
The dispensing device according to any one of <20> to <21>, wherein the droplet discharge unit discharges the droplet again to the same concave portion.
<23> The dispensing device according to any one of <20> to <22>, which is used for forming a tissue body.

  <1> to <11>, the particle counting device according to any one of <12> to <18>, the particle counting method according to <19>, and The dispensing device according to any one of <20> to <23> can solve the conventional problems and achieve the object of the present invention.

DESCRIPTION OF SYMBOLS 1, 1A, 1B, 1C, 1D, 1E Droplet formation apparatus 2 Dispensing apparatus 10 Droplet discharge means 14 Discharge port 20 Drive means 30 Light irradiation means 41 Light reception means 42 Light reception means 50 Particle counting means 200 Particles capable of emitting light 201 Liquid 210 Droplet L Light Lf Light emission

JP 2008-693 A

Claims (20)

A light irradiation means that includes particles capable of emitting light when irradiated with light, and irradiates the light to a plurality of droplets ejected from different positions;
A light receiving means for receiving light emitted from the particles irradiated with the light;
Particle counting means for counting the particles contained in the plurality of droplets based on the light emission received by the light receiving means;
A particle counting apparatus comprising:   The particle counter according to claim 1, further comprising two or more of the light receiving means, wherein each of the light receiving means receives light emitted from the particles from different directions. The light receiving means obtains information on the luminance value of the light emission received in a substantially normal direction of the light receiving surface and the shape of the light emission on the light receiving surface,
A first counting process in which the particle counting means counts the particles contained in the droplet based on the luminance value of the light emission; and the droplet based on the shape information on the light receiving surface of the light emission. The particle counting apparatus according to claim 2, wherein at least one of a second counting process for counting the contained particles is performed.   The particle counting device according to claim 3, wherein when the particle counting unit performs the first counting process and determines that the particles cannot be counted, the particle counting unit performs the second counting process and counts the particles.   The particle counting means calculates a radius of curvature based on the shape information of the light emission on the light receiving surface, and counts the number of circle centers when the radius of curvature within a predetermined range is calculated as the number of particles. The particle counting apparatus according to claim 3, wherein the second counting process is performed.   6. The device according to claim 2, wherein at least one of the two or more light receiving units is disposed such that a light receiving direction thereof is positioned substantially perpendicular to a light receiving direction of the other light receiving unit. The particle counter of description.   The particle counter according to any one of claims 2 to 6, wherein the light receiving means is arranged so that a light receiving direction thereof is positioned in a direction substantially orthogonal to a discharge direction of the droplets.   The particle counter according to any one of claims 1 to 7, wherein the light emitted from the light irradiation means is pulsed light.   The particle counter according to any one of claims 1 to 8, wherein the particles that can emit light when irradiated with light are cells.   The particle counter 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 cells stained with a fluorescent dye and cells capable of expressing a fluorescent protein. The particle counter according to any one of claims 1 to 10,
Droplet discharge means having a plurality of discharge ports as discharge start points;
A droplet forming apparatus comprising:   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 discharge ports is 2 or more and 100 or less per row. The droplet discharge means is
A liquid holding unit for holding a liquid containing particles capable of emitting light when irradiated with the light;
A plurality of discharge ports are formed, and a film-like member that discharges the liquid held in the liquid holding portion as droplets from the plurality of discharge ports by vibration,
The droplet forming apparatus according to claim 11, comprising:   The liquid droplet forming apparatus according to claim 14, wherein the liquid holding unit includes an atmosphere opening unit. A light irradiation step that includes particles capable of emitting light when irradiated with light, and irradiates the plurality of droplets discharged from different positions with the light;
A light receiving step for receiving light emitted from the particles irradiated with the light;
A particle counting step of counting the particles contained in the plurality of droplets based on the emitted light received in the light receiving step;
A particle counting method comprising: A droplet forming device according to any one of claims 11 to 15,
An object to be deposited on which a plurality of recesses to which the droplets ejected from the droplet ejection means are deposited; and
A dispensing device characterized by comprising:   The dispensing apparatus according to claim 17, further comprising a control unit that controls a relative positional relationship between the droplet discharge unit 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 zero,
The dispensing apparatus according to any one of claims 17 to 18, wherein the droplet discharge means discharges the droplet again to the same concave portion.   The dispensing apparatus according to any one of claims 17 to 19, which is used for forming a tissue body.