JP4146310B2 - Method for evaluating composition uniformity and apparatus for evaluating composition uniformity - Google Patents

Description

  The present invention relates to an evaluation method and an evaluation apparatus for composition uniformity in mixed droplets formed by mixing a plurality of raw material liquids having different compositions.

  Conventionally, for example, as described in Patent Document 1, a pulse voltage is applied between a raw material liquid stored for each of a plurality of capillaries and a substrate disposed facing the tip of each capillary. A method of forming a mixed droplet on a substrate by discharging and mixing a small amount of a raw material jet from the tip of each capillary is known. Here, the jet flow is a jet having a small diameter in which the raw material liquid interface is drawn from the capillary tip by applying a pulse voltage.

For example, as described in Patent Document 2, an apparatus that optically analyzes such a minute mixed droplet is known.
International Publication No. WO03 / 020418 Pamphlet JP 2002-055051 A

  In forming the minute mixed droplets as described above, it is necessary to evaluate whether or not each raw material liquid is uniformly mixed in the formed mixed droplets, that is, the uniformity of the composition of the mixed droplets. .

  However, a method and an apparatus for evaluating the uniformity of the composition of such minute mixed droplets are not known.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a composition uniformity evaluation method and an evaluation apparatus capable of evaluating the composition uniformity of minute mixed droplets. .

  The method for evaluating the uniformity of composition according to the present invention is the method for evaluating the uniformity of composition in a mixed droplet formed by mixing a plurality of raw material liquids having different compositions. An image acquisition step of collecting and image-forming light that reaches at an intensity corresponding to the amount of any one of the components to acquire image information about the mixed droplet, and acquiring the center position of the mixed droplet in the image information A center acquisition step, and a symmetry acquisition step of acquiring, in the image information, information relating to the symmetry of the intensity of light with respect to the center position of the mixed droplet.

  In addition, the apparatus for evaluating the uniformity of the composition according to the present invention provides a strength corresponding to the amount of any component in a mixed droplet formed from a mixed droplet formed by mixing a plurality of raw material liquids having different compositions. In the image information, the liquid mixture is obtained by condensing and merging the light arriving at the image acquisition means for acquiring the image information about the mixed droplet, the center acquisition means for acquiring the center position of the mixed droplet in the image information, Symmetry acquisition means for acquiring information on the symmetry of the intensity of light with respect to the center position of the droplet.

  According to these inventions, information on symmetry with respect to the center position of the light intensity distribution according to the amount of the above-mentioned component is acquired, and this information is considered as the distribution amount of the above-mentioned component around the center of the mixed droplet. Can do. If the above components are distributed with high symmetry around the center in the mixed droplet, it is considered that the composition uniformity in the mixed droplet is high. Therefore, based on the information about the symmetry around the center, mixing is performed. It is possible to easily and quickly evaluate the composition uniformity in the droplet.

  Here, the raw material liquid includes a component that absorbs light of a predetermined wavelength, and in the image acquisition process, the mixed droplet is irradiated with light of a predetermined wavelength, and the transmitted light that has passed through the mixed droplet is collected. It is preferable to form an image and acquire image information.

  According to this, in the image information, as the amount of the component that absorbs the predetermined wavelength in the mixed droplet is larger, the intensity of the light becomes weaker. The uniformity of composition in the droplet can be obtained with higher accuracy.

  In addition, the raw material liquid contains a fluorescent dye that emits fluorescence when irradiated with excitation light. In the image acquisition process, the mixed droplets are irradiated with excitation light, and the fluorescence generated and reached by irradiation of the excitation light is emitted. Image information may be acquired by focusing and forming an image.

  According to this, in the image information, as the amount of the fluorescent dye in the mixed droplet is larger, the intensity of light becomes stronger. Therefore, the distribution amount of the fluorescent dye is appropriately grasped in the image information, and the composition in the mixed droplet is determined. Can be obtained more accurately.

  In addition, when acquiring image information by fluorescence, light is further applied to the mixed droplet, and the transmitted light transmitted through the mixed droplet is condensed to form an image. Before performing the symmetry acquisition step, it is preferable to include a correction step of correcting the fluorescence image information based on the transmitted light image information, whereby the uniformity can be acquired with higher accuracy.

  The center acquisition step acquires the outer edge shape of the mixed droplet in the image information, approximates the outer edge shape to an ellipse including a perfect circle, and acquires the center position of the ellipse including the perfect circle as the center position of the mixed droplet. It is preferable.

  The center acquisition step may acquire a region occupied by the mixed droplet in the image information, and acquire the center of gravity of the mixed droplet region as the center position of the mixed droplet.

  In the center acquisition step, the center of gravity of the light intensity distribution may be acquired in the image information, and the center of gravity position of the light intensity distribution may be acquired as the center position of the mixed droplet.

  According to these, the center position of the mixed droplet in the image information can be acquired easily and suitably.

  Further, in the evaluation method of the uniformity of the composition described above, the mixed droplet applies a pulse voltage between the raw material liquid stored for each of the plurality of capillaries and the substrate disposed facing the tip of the capillary, It is preferably formed on the substrate by injecting the raw material liquid from the tip of each capillary.

  Further, in the apparatus for evaluating the uniformity of the composition, a raw material liquid injection section having a plurality of capillaries each storing a raw material liquid for forming mixed droplets is disposed opposite the tip of the capillary, A voltage applying device for applying a pulse voltage between a substrate on which a mixed droplet formed from a raw material liquid ejected from the tip of each capillary is placed, and each of the raw material liquid stored in each capillary and the substrate It is preferable to further include

  According to these, minute mixed droplets can be suitably formed, and the uniformity of the composition of the mixed droplets thus formed can be measured quickly.

  Moreover, in the evaluation apparatus for the uniformity of the composition, an electrode is provided on the outer periphery of at least one capillary out of a plurality of capillaries, and this evaluation apparatus further applies a potential higher than the potential of the raw material liquid to the electrode. It is preferable to include a control device that controls the voltage application device.

  In this case, if the voltage application device is controlled by the control device so that a potential equal to or higher than the potential of the raw material liquid is applied to the electrode, the lines of electric force are more concentrated immediately below the nozzle. For this reason, it is possible to accurately arrange the raw material liquid at a desired position on the droplet formation object. For this reason, when discharging a raw material liquid toward a to-be-droplet formation object after that, it can be made to mix with the droplet consisting of a raw material liquid exactly. Further, since the mixing of the raw material liquids is not performed before being discharged from the nozzles but after the discharge, the quality of the raw material liquids does not change at each nozzle.

  According to the present invention, the uniformity of the composition of minute mixed droplets can be easily and quickly evaluated. As a result, by forming mixed droplets under various conditions and evaluating the uniformity of each mixed droplet, it is possible to easily grasp the droplet formation conditions necessary for good mixing. .

Hereinafter, preferred embodiments of a method and an apparatus for evaluating the uniformity of composition of mixed droplets according to the present invention will be described in detail with reference to the accompanying drawings. In the description of the drawings, the same or corresponding elements are denoted by the same reference numerals, and redundant description is omitted.
(First embodiment)
First, the composition uniformity evaluation apparatus according to the present embodiment will be described with reference to FIG. The composition evaluation device 100 for the composition uniformity of mixed droplets is roughly divided into a mixed droplet formation unit 110 and a mixed droplet uniformity evaluation unit 120.

  The mixed droplet forming unit 110 includes a raw material liquid injection unit 105 having a plurality of capillaries 101, 102, and 103 each storing different raw material liquids, and each of the raw material liquids in the capillaries 101, 102, and 103 and the electric stage 3. And a voltage applying device 107 that applies a voltage to the light-transmitting substrate 2 placed thereon.

  The tips of the capillaries 101, 102, 103 of the raw material liquid injection unit 105 are made to face the substrate 2 placed on the electric stage 3. The raw material liquid injection unit 105 includes an electrode 104 in each capillary 101, 102, 103 made of glass or the like as shown in FIG. 2, and this electrode 104 is connected to the raw material liquid 8a in the capillaries 101 to 103. Each comes into contact.

  The voltage application device 107 applies a predetermined pulse voltage between the electric stage 3 on which the substrate 2 is placed and each electrode 104. Thereby, a pulse voltage is applied between the substrate 2 on the electric stage 3 and each electrode liquid 8a, and the raw material liquid 8a in the capillaries 101 to 103 is respectively supplied to the substrate 2 as a raw material liquid jet flow R at an arbitrary timing. It is injected towards. The injected raw material liquid jet streams R merge with each other in the air or merge with each other on the substrate to form mixed droplets M.

  Here, each raw material liquid that forms each raw material liquid jet stream R in advance contains fluorescent substances that emit fluorescence having different wavelengths when irradiated with excitation light.

  On the other hand, referring back to FIG. 1, the mixed droplet uniformity evaluation unit 120 transmits the electric stage 3 on which the substrate 2 is placed, the electric stage control unit 108 that controls the movement of the electric stage 3, and the transmission from below the substrate 2. A transmitted light source 4 for irradiating white light for illumination to the mixed droplet M and a mixed mirror M on the substrate 2 having a half mirror 6 are irradiated with excitation light for fluorescence emission from above. A light source for fluorescence excitation 7, an objective lens 5 that focuses and radiates light emitted upward from the mixed droplet M of the substrate 2, and light of a desired wavelength among the light condensed by the objective lens 5 Is selectively transmitted, and a field image including the mixed droplet M imaged by the variable motor filter 8 is detected to obtain image data (image information) including the intensity of the light. Camera 9 and image data acquired by camera 9 An image processing apparatus 10 to sense, and a control unit 12 for controlling the mixture droplets uniformity evaluation unit 120. Here, the objective lens 5, the fluorescence excitation light source 7, the variable electric filter 8, and the camera 9 constitute the image acquisition means 115.

  The electric stage 3 moves the substrate 2 in the horizontal direction, and the position where the mixed droplet M formed on the substrate 2 can be observed by the mixed droplet uniformity evaluation unit 120, specifically, the objective lens 5. Move to the same position on the optical axis.

  The transmitted light source 4 emits white light for transmitted illumination from the back side of the mixed droplet M, and transmits the mixed droplet M and the substrate 2 to generate upward transmitted light.

  The fluorescence excitation light source 7 irradiates the mixed droplet M on the substrate 2 with excitation light via the half mirror 6 and the objective lens 5. This excitation light is light having a wavelength for exciting each fluorescent component of the raw material liquid included in the mixed droplet M. When the mixed droplet M is irradiated with excitation light, the mixed droplet M generates fluorescence having an intensity corresponding to the amount of the fluorescent component. That is, fluorescence is generated at a high intensity from a portion where the amount of the fluorescent component is large. Here, the fluorescence excitation light source 7 can individually irradiate wavelengths for exciting each fluorescent substance in the raw material liquid.

  The objective lens 5 collects the transmitted light transmitted through the mixed droplet M and the substrate 2 and the fluorescence generated from the mixed droplet M, and passes on the image sensor of the camera 9 via the half mirror 6 and the variable electric filter 8. This transmitted light image or fluorescent image is formed.

  The variable electric filter 8 selectively transmits light having a predetermined wavelength from light incident from the objective lens 5, and the predetermined light can be appropriately selected by the control device 12. The selectable wavelength corresponds to the fluorescence wavelength of the fluorescence component and the wavelength of the white light source of the transmitted light source 4.

  The camera 9 collects the mixed droplet M and its surrounding substrate by detecting a transmitted light image or a fluorescent image focused by the objective lens 5 and transmitted through the half mirror 6 and the variable electric filter 8 to form an image. 2 and transmitted light image data and fluorescence image data of a predetermined field of view. These image data are image data obtained by photographing the inside of the field of view including the mixed droplet M, the substrate 2 and the like, and each pixel in each image data includes information on the intensity of transmitted light or fluorescence reached from each part in the field of view. Contains.

  Here, the camera 9 transmits transmitted light image data obtained when the mixed droplet M is irradiated with only white light for transmitted illumination, and fluorescent image data obtained when the mixed droplet M is irradiated with only the excitation light. Are obtained separately for one mixed droplet M.

  Next, a detailed configuration of the image processing apparatus 10 is shown as a block diagram in FIG. The image processing apparatus 10 includes an image data acquisition unit 50 that acquires fluorescent image data and transmitted light image data from the camera 9, an outline extraction unit 52 that extracts an outline of the mixed droplet M from the transmitted light image data, and an extraction A background noise removing unit 54 for removing background noise from the fluorescence image data, a leakage fluorescence removing unit 56 for removing leakage fluorescence from the fluorescence image data from which the background noise has been removed, and a leakage A center acquisition unit 58 for acquiring the center position of the mixed droplet M in the fluorescence image data from which fluorescence has been removed, and symmetry acquisition for acquiring an index relating to the symmetry of the fluorescence intensity around the acquired center position in the fluorescence image data. This is a computer device that exhibits the same functions as the unit 60 and the display unit 70 that displays the obtained results. Here, the image processing is performed for each combination of transmitted light image data and fluorescence image data acquired for one mixed droplet M.

  The contour extraction unit 52 extracts the contour line of the mixed droplet M based on the transmitted light image data. Here, for example, the contour line can be acquired by binarizing the transmitted light image data and taking differentiation between pixels.

  The background noise removing unit 54 refers to the contour line extracted by the contour extracting unit 52 and regards the fluorescence image data as background noise in the fluorescence image data by assuming that there is no fluorescent substance outside the contour line. Remove. Specifically, the fluorescence intensity of the pixels on the contour line in the fluorescence image data is acquired, and background noise of the fluorescence image is removed based on the fluorescence intensity of the pixels on the contour line. For example, the background noise can be removed by obtaining the average value of the fluorescence intensity of the pixels on the contour line and subtracting this average value from the data of the light intensity of all the pixels of the fluorescence image data.

  The leakage fluorescence removing unit 56 removes leakage fluorescence from the fluorescence image data from which background noise has been removed. Usually, since the fluorescence has a wide spectrum, even if it is other fluorescence having a center wavelength different from the transmission wavelength range of the variable electric filter 8, the bottom part of the spectrum is slightly transmitted through the variable electric filter 8. Therefore, it is necessary to correct the fluorescence image data by removing such leakage fluorescence. A typical correction method is as follows. For example, when two kinds of fluorescent dye solutions are mixed, the measured values of the fluorescence intensity acquired through the filter for each fluorescence are set as f and g, respectively. At this time, if the true fluorescence intensity for each fluorescence is F and G, f and g can be expressed as f = F + aG and g = G + bF. Leakage fluorescence correction can be performed by solving these simultaneous equations and obtaining F and G using these two equations. Here, aG and bF represent leaking fluorescence, and a and b are the transmittances of the filters for different fluorescence. Here, a and b are constants measured in advance before the experiment.

  The center acquisition unit (center acquisition means) 58 acquires the center coordinates of the mixed droplet M in the fluorescence image data in which leakage fluorescence is corrected. Specifically, for example, the following three methods can be taken.

  The first method acquires the outer edge shape of the mixed droplet M in the fluorescence image data, approximates the outer edge shape to an ellipse including a perfect circle, and sets the center position of the ellipse including the true circle as the center of the mixed droplet M. It is a coordinate. The second method is to obtain the centroid position of the fluorescence intensity distribution in the fluorescence image data and use this centroid as the center coordinate of the mixed droplet M. In the third method, the outer edge shape of the mixed droplet M is acquired in the fluorescence image data, the area centroid of the region of the outer edge shape is acquired, and the area centroid is used as the center coordinate of the mixed droplet M.

  The symmetry acquisition unit (symmetry acquisition means) 60 evaluates the symmetry of the fluorescence intensity around the center coordinates of the mixed droplet M in the fluorescence image data.

Specifically, as shown in FIG. 4A, when polar coordinates (r, θ) having the origin at the center coordinate of the mixed liquid droplet M are set in the obtained fluorescent image, on the r-axis with respect to an arbitrary θ. The fluorescence intensity distribution curve is as shown in FIG. That is, since the mixed droplet M is usually thickest at the central portion, the intensity of fluorescence reaching from the central portion is often the strongest, and in this case, a convex convex distribution curve is obtained. Here, the distribution curve is divided into two regions by a vertical axis line where r = 0, the area formed by the distribution curve on one side and the X axis is formed by S _R , and the distribution curve on the other side and the X axis are formed. Let S _L be the area. The difference or ratio between S _L and S _R indicates symmetry about the central axis of the fluorescence intensity distribution. Here, with respect to θ (0 ≦ θ <π), a function δ (θ) [%] as a degree of uniformity is defined as in equation (1).

（１）
そして、このδを、θ＝０〜πにおいて積分して平均することにより、（２）式のように均一度の指標となる対称度Δをそれぞれの蛍光画像データに対して求めることができる。

(1)
Then, by integrating and averaging δ at θ = 0 to π, a symmetry Δ that is an index of uniformity can be obtained for each fluorescent image data as shown in equation (2).

（２）
ここで、対称度Δ＝０であれば、中心座標周りに対称な濃度分布が混合液滴Ｍ内に形成されていることを示しており、これは、混合度が極めて高くほぼ完全に混合されていることを示す。一方、対称度Δ＞０であれば、中心周りに非対称な濃度分布が混合液滴Ｍ内に形成されていることを示し、特に、対称度Δが大きくなればなるほど不均一度が高い、すなわち、混合が不十分であることを示している。

(2)
Here, if the degree of symmetry Δ = 0, it indicates that a symmetric concentration distribution around the center coordinate is formed in the mixed droplet M, which is extremely high in mixing and almost completely mixed. Indicates that On the other hand, if the degree of symmetry Δ> 0, this indicates that an asymmetric concentration distribution around the center is formed in the mixed droplet M, and in particular, the higher the degree of symmetry Δ, the higher the degree of non-uniformity. , Indicating insufficient mixing.

  The display unit 70 displays the image data acquired by the image data acquisition unit 50 and the symmetry degree Δ acquired by the symmetry acquisition unit 60 to the operator using a display or the like.

  Subsequently, returning to FIG. 1, the control device 12 obtains good transmitted light image data / fluorescence image data, the transmitted light source 4, the fluorescence excitation light source 7, the variable electric filter 8, the camera 9, and the electric stage. The control unit 108, the image processing device 10, and the voltage application device 107 are controlled.

  Next, the operation of the composition uniformity evaluation apparatus 100 according to the present embodiment will be described with reference to the flowchart of FIG.

  In advance, the variable electric filter 8 is set in a state capable of transmitting light of all wavelengths.

  First, the mixed droplet M is formed on the substrate 2 (step S201). Specifically, the voltage application device 107 applies a pulse voltage between each raw material liquid 8a in the capillaries 101, 102, and 103 and the substrate 2, and the raw material liquid jet flow R of each raw material liquid from the capillaries 101 to 103. Are ejected onto the substrate 2 to form a mixed droplet M in which the raw material liquid jet stream R is mixed on the substrate 2. More specifically, as shown in FIG. 2, after application of a pulse voltage, the raw material liquid 8a is drawn out from the capillaries 101, 102, and 103 by electrostatic attraction, and a Taylor Cone 16 is formed. A fixed amount of raw material liquid is discharged as a jet stream R, and as a result, mixed droplets M are formed on the substrate 2. Here, the raw material liquid jet streams R may be injected almost simultaneously from the capillaries 101 to 103 and mixed with each other in the air before reaching the substrate 2. Alternatively, each raw material liquid jet stream R may be individually injected toward the substrate 2 and mixed on the substrate 2. Here, a plurality of mixed droplets M are formed on the substrate 2. At this time, each raw material liquid contains fluorescent substances that emit fluorescence having different wavelengths when irradiated with excitation light.

  Next, the electric stage 3 is moved so that one mixed droplet M on the substrate 2 enters a predetermined position in the field of view of the objective lens 5 (step 202). Next, using the transmitted light source 4, the mixed droplet M is irradiated with white light from the back surface of the substrate 2 (step S 203), and the transmitted light from the mixed droplet M is detected by the camera 9. Thus, transmitted light image data is acquired (step S204).

  Subsequently, after the transmitted light source 4 is quenched, the fluorescence excitation light source 7 emits light of a wavelength for exciting the fluorescent material of each raw material liquid contained in the mixed droplet M, and the variable electric filter 8. Is set so that only the excitation light is transmitted (step S205). Then, fluorescence emitted from the mixed droplet M with an intensity corresponding to the amount of the fluorescent component in each portion is detected by the camera 9 to acquire fluorescent image data (step S206).

  Next, in step S207, it is determined whether or not fluorescent image data has been acquired for the fluorescent substances corresponding to all the raw material liquids in one mixed droplet M, and there is fluorescent image data relating to unacquired fluorescent substances. Then, returning to step S205 again, different excitation light is generated from the fluorescence excitation light source 7, and the variable electric filter 8 is reset so that only the fluorescence corresponding to the excitation light is transmitted, and the step again. In S206, other fluorescent image data is acquired. In this way, while the transmitted light image data and each fluorescence image data are acquired for one mixed droplet M, the mixed droplet M is fixed in the field of view of the camera 9, so that the transmitted light image data And the position of the mixed droplet M in the fluorescence image data is the same.

  On the other hand, when the fluorescence image data is acquired for all the fluorescence components in one mixed droplet M, in step S208, the combination of the transmitted light image data and the fluorescence image data is obtained for all the mixed droplets M on the substrate 2. It is determined whether or not it has been acquired, and if there is a mixed droplet M for which data has not been acquired, the process returns to step S202 to acquire each image data of another mixed droplet M again.

  On the other hand, if it is determined in step S208 that a combination of transmitted light image data and fluorescence image data of all the mixed droplets M on the substrate 2 has been acquired, the process proceeds to an image processing step after step S209.

  First, in step S209, the contour line of the mixed droplet M is extracted from the transmitted light image data in the combination of the image data concerning one mixed droplet M. Next, the process proceeds to step S210, and background noise is removed from each fluorescent image data related to the single mixed droplet M by using the contour line obtained in step S209. Subsequently, in step S211, leakage fluorescence is removed for each fluorescence image data from which background noise has been removed.

  Then, the process proceeds to step S212, where it is determined whether or not the fluorescence image data has been corrected for all the mixed droplets M. If there is uncorrected fluorescence image data, the process returns to step S209, and again the fluorescence image data. Compensate for.

  On the other hand, if the fluorescence image has been corrected for all the mixed droplets M, the process proceeds to step S213, and the selection of the method for determining the center of the mixed droplet M in the fluorescence image data is input from the operator. Here, the operator can select one of the three methods for determining the center position of the mixed droplet M described above.

  Then, when the determination method of the center position of the mixed droplet M in the fluorescence image data is selected, in step S214, the fluorescence of one mixed droplet M is determined according to the determination method of the center position of the selected mixed droplet M. The center coordinates of the mixed droplet M in the image data are obtained.

  Subsequently, in step S215, the center coordinates acquired in step S214 are referred to for each fluorescent image data relating to the single mixed droplet M, and the symmetry degree around the center is determined for the fluorescence intensity data of the pixels around the center coordinates. Find Δ. In step S216, it is determined whether or not the degree of symmetry Δ has been obtained for the fluorescence image data relating to all the mixed droplets M, and there is fluorescence image data relating to the mixed droplets M for which the degree of symmetry Δ has not been acquired. Then, the process returns to step S214, and the degree of symmetry Δ is acquired for each fluorescence image data of each mixed droplet M. On the other hand, when the symmetry Δ in each fluorescence image is acquired for all the mixed droplets M, the process proceeds to step S217, and statistical processing of the symmetry Δ related to each mixed droplet M obtained in this way is performed. I do. For example, it is possible to acquire the average value of the symmetry Δ for each fluorescent component or to acquire the average value of the symmetry Δ of all the fluorescent components. As described above, the degree of symmetry Δ indicates that the larger the number is, the more nonuniformity is, that is, the mixed state is poor.

  As described above, according to the apparatus and method for evaluating the uniformity of the composition according to the present embodiment, the light reaching the mixed droplet M with the intensity corresponding to the amount of the component of the mixed droplet M is condensed to form an image. The fluorescence image data for the mixed droplet M is acquired, the center position of the mixed droplet M in the fluorescence image data is further acquired, and the symmetry Δ of the light intensity around the center position of the mixed droplet in the fluorescence image data is obtained. Have acquired.

  This degree of symmetry Δ can be considered as the symmetry of the distribution amount of the fluorescent substance around the center in the mixed droplet. If each fluorescent substance is distributed with high symmetry around the center in the mixed droplet, it is considered that the composition uniformity in the mixed droplet is high. Therefore, based on this symmetry Δ, The uniformity of composition can be easily and quickly evaluated. In particular, since the mixed droplet M formed as in the present embodiment has a very small volume, it is easily affected by evaporation, and the uniformity of the composition of the mixed droplet M can be evaluated in a short time after formation. The uniformity can be measured with high accuracy.

  Each raw material liquid supplied to the mixed droplet M includes a fluorescent dye that emits fluorescence when irradiated with excitation light. The mixed droplet M is irradiated with excitation light and irradiated with excitation light. Fluorescence image data of a visual field including mixed droplets is acquired by detecting the generated fluorescence.

  As a result, the intensity of light increases as the amount of the fluorescent dye in the mixed droplet M increases. Therefore, the distribution degree of each fluorescent component in the mixed droplet M is suitably grasped in the fluorescence image information, and the mixed droplet M The uniformity of the composition is obtained with higher accuracy.

  Further, before the mixed droplet M is irradiated with light, the transmitted light transmitted through the mixed droplet M is detected to obtain transmitted light image data of the visual field including the mixed droplet M, and before the degree of symmetry Δ is acquired. In addition, the fluorescence image information is corrected based on the transmitted light image data. Therefore, the degree of symmetry Δ can be acquired with higher accuracy based on the fluorescence image data.

  Further, the outer edge shape of the mixed droplet M is acquired in the image data, the outer edge shape is approximated to an ellipse including a perfect circle, and the center position of the ellipse including the perfect circle is set as the center position of the mixed droplet M, or the image data The area occupied by the mixed droplet M is acquired as the center position of the mixed droplet M as the center position of the mixed droplet M, or the center of the intensity distribution of light in the image data is the center of the mixed droplet M. Since it is acquired as the position, the center position of the mixed droplet M in the image data can be acquired easily and suitably. For this reason, the symmetry degree Δ of the mixed droplet is obtained with higher accuracy.

  Such a technique is particularly effective in fields such as combinatorial chemistry and drug screening using minute mixed droplets.

  Next, an example of uniformity measurement according to the present embodiment will be described.

First, a first solution containing fluorescein and a second solution containing rhodamine were prepared. Here, the first solution contains fluorescein at a concentration of 1.25 × 10 ⁻⁴ mol / L and sodium chloride at a concentration of 1.1 × 10 ⁻² mol / L in the solvent. This solvent consists of 95 wt% glycerin, 2.5 wt% water, 2.5 wt% ethanol. At this time, the viscosity coefficient of the first solution was 906 × 10 ⁻³ Pa · s.

On the other hand, the second solution, in a solvent, but at a concentration of Rhodamine ^{B 1.25 × 10 -4 mol / L} , sodium chloride 1.1 × 10 ^-2 mol / L. Here, the solvent is composed of 95 wt% glycerin and 5 wt% water. The viscosity coefficient of the second solution was 616 × 10 ⁻³ Pa · s.

In addition, such a 1st solution and a 2nd solution are very high in viscosity so that it is clear compared with the water whose viscosity coefficient is about 1 * 10 < ^-3 > Pa * s, and it is quite difficult to mix uniformly. Was expected. Since the first and second solutions mainly composed of glycerin have a very low evaporation rate, it is unlikely that the mixed droplets evaporate and the shape, concentration distribution, and the like change.

  Subsequently, the first solution and the second solution are separately introduced into the capillaries 101 and 102, a voltage is applied between the substrate 2 and the first solution in the capillary 101, and the first solution is electrostatically attracted. A raw material liquid jet stream R was ejected toward the substrate 2 to form a raw material droplet of the first solution on the substrate 2. Further, a voltage was applied between the second solution in the capillary 102 and the substrate 2, and the second solution was injected as a raw material liquid jet stream R onto the raw material droplets of the first solution on the substrate 2. As a result, a mixed droplet MA in which the first solution and the second solution were mixed on the substrate 2 was formed.

  Specifically, a mixed droplet MA having a volume of the first solution droplet ejected from the capillary 101 of 14 pL and a volume of the second solution of 2.7 pL was formed. Furthermore, a mixed droplet MB was formed in which the volume of the first solution droplet was 14 pL and the volume of the second solution was 0.7 pL. At this time, a quartz substrate with an ITO (Indium-Tin Oxide) thin film was used as the substrate 2. ITO functions as an electrode when electrostatically attracting droplets.

  The mixed droplets MA and MB thus obtained were evaluated for composition uniformity using the above-described composition uniformity evaluation apparatus.

  The transmitted light image of the mixed droplet MA thus obtained is shown in FIG. 6 (a), the fluorescence image of fluorescein is shown in FIG. 6 (b), and the fluorescence image of rhodamine B is shown in FIG. 6 (c). . 6 (d) shows a fluorescence intensity profile along the horizontal line in the fluorescence image of FIG. 6 (b), and FIG. 6 (e) shows a fluorescence intensity profile along the horizontal line of the fluorescence image of FIG. 6 (c). Shown in

  Also, the transmitted light image of the mixed droplet MB thus obtained is shown in FIG. 7A, the fluorescence image of fluorescein in FIG. 7B, and the fluorescence image of rhodamine B in FIG. 7C. Each is shown. 7 (d) shows a fluorescence intensity profile along the horizontal line in the fluorescence image of FIG. 7 (b), and FIG. 7 (e) shows a fluorescence intensity profile along the horizontal line of the fluorescence image of FIG. 7 (c). Shown in In FIGS. 6 and 7, the fluorescence image and the fluorescence intensity profile indicate the mixed droplets MA and MB after the leakage fluorescence correction, respectively.

  Here, the center of gravity of the mixed droplets MA and MB in each fluorescence image is the center of gravity of the fluorescence distribution of the fluorescence image of fluorescein.

  Regarding the mixed droplet MA, both the rhodamine B profile and the fluorescein profile show good objectivity, and both rhodamine B and fluorescein have almost symmetrical fluorescence intensity distributions around the center, and the fluorescein symmetry Δ = 0. The degree of symmetry of Rhodamine B was Δ = 0.015. The mixed state was judged to be relatively good.

On the other hand, with respect to the mixed droplet MB, in particular, the symmetry with respect to rhodamine B was poor, and the symmetry of fluorescein Δ = 0.015, whereas the symmetry of rhodamine B was Δ = 0.045. This indicates that in the mixed droplet MB, the component of rhodamine B is present more unevenly around the center than in the mixed droplet MA, that is, the mixed droplet MB has a lower uniformity of composition. It was confirmed that the uniformity of the composition of the mixed droplets can be easily and suitably quantified.
(Second Embodiment)
Next, an evaluation apparatus 200 for the composition uniformity of mixed droplets according to a second embodiment will be described with reference to FIG. The composition uniformity evaluation apparatus 200 according to the present embodiment is different from the composition uniformity evaluation apparatus according to the first embodiment in that a fluorescence excitation light source 7 and a half mirror for acquiring fluorescence image data are used. 6 and instead of the variable electric filter 8, the transmitted white light source 21 and the white light from the transmitted white light source 21 transmit only light in a predetermined wavelength range to transmit light in the wavelength range. And a transmitted light spectroscope 22 that irradiates the mixed droplet M from the back surface of the substrate 2. Here, the objective lens 5, the camera 9, the transmitted light spectroscope 22, and the transmitted light white light source 21 constitute an image acquisition unit 115.

  Here, the light in the predetermined wavelength range is monochromatic light corresponding to the absorption wavelength of each raw material liquid component contained in the mixed droplet M.

  Corresponding to this, the camera 9 detects the transmitted light image data including the mixed droplet M by detecting the transmitted light image that is transmitted through the mixed droplet M and collected by the objective lens 5 and formed. get.

  Further, in response to this, as shown in FIG. 9, in the image processing apparatus 10, the image data acquisition unit 50 acquires the transmitted light image data from the camera 9, and the contour line extraction unit 52 determines the image from the transmitted light image data. The contour of the mixed droplet M is acquired, and the absorbance distribution acquisition unit 150 uses the transmitted light intensity in the region outside the contour of the mixed droplet M as the reference light (baseline when there is no mixed droplet M). Obtain distribution image data. Further, in the image processing apparatus 10, the center of the mixed droplet M is determined by the above-described center acquisition unit 58 with respect to the absorbance distribution data acquired in this way, and the symmetry Δ is obtained by the symmetry acquisition unit 60. The composition uniformity is quantified, and the result is displayed on the display unit 70.

  Here, the center acquisition unit 58 acquires the center coordinates of the mixed droplet M in the transmitted light image data of each monochromatic light. Specifically, for example, the following three methods can be taken.

  First, the contour line acquired by the contour line extraction unit 52 can be approximated to an ellipse including a perfect circle, and the center position of the ellipse including the true circle can be used as the center coordinates of the mixed droplet M. Second, the area surrounded by the contour line can be acquired, the area centroid of this area can be acquired, and the area centroid can be used as the center coordinates of the mixed droplet M. Third, in the absorption distribution data, the center of gravity of the absorbance distribution can be obtained, and this center of gravity can be used as the center coordinates of the droplet.

  Note that the transmitted light white light source 21, the transmitted light spectroscope 22, the camera 9, and the image processing device 10 are controlled by the control device 12 in order to obtain good transmitted image data.

  Next, the operation of the apparatus for evaluating the uniformity of the composition of the mixed droplet M will be described with reference to the flowchart of FIG.

  First, a plurality of mixed droplets M are formed on the substrate 2 as in the first embodiment (step S201). Here, each raw material liquid stored in each capillary of the raw material liquid injection unit 105 and supplied to the mixed droplet M as the raw material liquid jet stream R includes substances that absorb light of different wavelengths.

  Next, in the mixed droplet M, in order to identify the distribution of the components of the mixed droplet M by the absorbance, the operator designates a specific absorption wavelength for each raw material liquid (step S401). Next, the electric stage 3 is moved so that the mixed droplet M in the substrate 2 enters a predetermined position in the field of view of the objective lens 5 (step S202). Next, after the light from the white light source for transmitted light 21 is converted into monochromatic light corresponding to the above-described intrinsic absorption wavelength by the transmitted light spectroscope 22 (step S404), the mixed droplet M is applied to the mixed droplet M from the back surface of the substrate 2. Then, the monochromatic light of that wavelength is irradiated (step S405), the transmitted light from the mixed droplet M is detected by the camera 9, and transmitted light image data is acquired (step S406).

  In step S407, it is determined whether or not transmitted light image data having a wavelength corresponding to the light absorption component of all raw material liquids has been acquired for the mixed droplet M, and there is transmitted light image data relating to an unacquired wavelength. For example, the process returns to step S404 again, monochromatic light having a different wavelength is selected by the transmitted light spectroscope 22, and transmitted light image data is acquired again in step S405. In this way, while acquiring the transmitted light image data with monochromatic light of each wavelength for one mixed droplet M, the mixed droplet M is fixed in the field of view of the camera 9, so that for each wavelength. The positions of the mixed droplets M in the transmitted light image data are the same.

  On the other hand, when the transmitted light image data is acquired for all the wavelengths in one mixed droplet M, it is determined in step S408 whether the transmitted light image data is acquired for all the mixed droplets M on the substrate 2. If it is determined that there is a mixed droplet M for which data has not been acquired, the process returns to step 202 to acquire data for another mixed droplet M again.

  On the other hand, if it is determined in step S408 that the image data of all the mixed droplets M has been acquired, the process proceeds to an image processing process in step S409 and subsequent steps.

  First, in step S409, the type of wavelength corresponding to the transmitted light image data having the clearest outline of the mixed droplet M is selected from the transmitted light image data of each wavelength related to one mixed droplet M. Is selected (step S409). Next, transmitted light image data of each wavelength related to one mixed droplet M is selected (step S410), and transmitted light image data related to the wavelength selected in step S409 is selected from the transmitted light image data of each wavelength. Then, the outline of the mixed droplet M is acquired (step S411). Next, one transmitted light image data is selected from the transmitted light image data corresponding to each wavelength (step S412), and the intensity of the light outside the outline of the mixed droplet M in the transmitted light image data is referred to as the reference light. As described above, absorbance distribution image data is created (step S413).

  Then, the process proceeds to step S414, where it is determined whether or not the absorbance distribution image data has been formed for the transmitted light image data of all wavelengths. If there is transmitted light image data relating to unacquired wavelengths, the process returns to step S412. Thus, absorbance distribution image data is created for each wavelength.

  On the other hand, if the absorbance distribution image data has been acquired for all wavelengths, the process proceeds to step S415, and it is determined whether or not the absorbance distribution image has been acquired for all the mixed droplets M, and the unacquired mixing is performed. If there is a droplet M, the process returns to step S410 to acquire absorbance distribution image data for each wavelength for the remaining mixed droplet M.

  On the other hand, if the absorbance distribution image data is obtained for all the mixed droplets M, the process proceeds to step S416, and selection of a method for determining the center of the mixed droplet M is input from the operator. Here, the operator can select one of the three methods for determining the center position of the droplet.

  Subsequently, in step S417, using the selected method, the central coordinates of the absorbance distribution image data are obtained for each mixed droplet M (step S417), and then each absorbance distribution for each mixed droplet M is obtained. For each image data, a degree of symmetry Δ related to the uniformity of absorbance around the center is obtained (step S418). In step S419, it is determined whether or not this degree of symmetry Δ has been acquired for all the mixed droplets M. If there is an unacquired mixed droplet M, the process returns to step S417 to return to another mixed droplet. On the other hand, if the symmetry degree Δ is calculated for all the mixed droplets, the process proceeds to step S420, and the mixed state evaluation symmetry degree Δ is statistically processed for all the mixed droplets M. Then (step 420), a comprehensive evaluation is made as to whether or not the composition in the mixed droplet M is uniform.

According to the present embodiment, the raw material liquid includes a component that absorbs light having a predetermined wavelength, and the mixed droplet M is irradiated with light having the predetermined wavelength, and the transmitted light transmitted through the mixed droplet M is transmitted. Absorption image data (corresponding to image information) of the visual field including the mixed droplet M is detected and acquired. In the absorption image data, the intensity of light becomes weaker as the amount of the component that absorbs the predetermined wavelength in the mixed droplet M becomes larger. Therefore, the distribution amount of this component is suitably grasped in the absorption image data and mixed. The uniformity of the composition in the droplet M can be obtained with high accuracy as in the first embodiment, and the same effects as in the first embodiment can be achieved.
(Third embodiment)
Next, an apparatus for evaluating the uniformity of the composition of mixed droplets according to the third embodiment will be described. The evaluation value of the uniformity of composition according to this embodiment is different from that of the first embodiment only in the capillaries 101, 102, 103 and the control device 12 of the raw material liquid injection unit 105 in the mixed droplet forming unit 110. Therefore, other explanation is omitted.

  As shown in FIG. 11, in this embodiment, the capillaries 101, 102, and 103 further include external electrodes 320 on the outer periphery.

  Here, the constituent material of the external electrode 320 is not particularly limited as long as it has conductivity, but as such a constituent material, gold or platinum is preferable for the purpose of preventing corrosion. The external electrode 320 can be formed by evaporating the above constituent material on the tip of the capillary 102, for example.

  Further, in the present embodiment, the control device 12 forms, for example, a voltage higher than the pulse voltage applied between the raw material liquid 8 a and the substrate 2 between the electrode 320 and the electric stage 3 when forming the mixed droplet M. The voltage application device 107 is controlled to apply the voltage.

  Then, the electrostatic induction charge 321 appearing at the tip of the external electrode 320 biases the charge distribution of the electrostatic induction charge 361 on the surface of the diluent so as to be the highest at the center of the nozzle. That is, a large electrostatic force acts between the center of the raw material liquid surface and the electric stage 3. As a result, the Taylor cone 16 remains in the inner diameter portion of the nozzle end surface, and its shape is deformed more sharply. This is a result of the electric lines of force being concentrated at the center of the nozzle. For this reason, the position where the droplet M is formed can be made extremely stable. In other words, the droplet M can be more accurately formed at a desired position on the substrate 2.

  Then, once the droplet M is formed on the substrate 2, in this embodiment, the raw material liquid can be accurately injected into the droplet M from another capillary. For example, the mixed droplet M having a desired concentration is desired. It can be accurately formed at the position.

  In addition, according to the present embodiment, although the Taylor cone 16 is formed, it remains at the inner diameter portion of the capillary 102 and the like, so that the tip end portion becomes sharp, and the liquid is easily cut off during ejection. For this reason, the distance between the raw material liquid 8a and the electric stage 3 can be reduced, and it can be driven with a relatively small voltage. Due to this effect, there is no fear of discharge between the raw material liquid 8a and the electric stage 3, and the reliability of the mixed droplet formation unit 110 can be improved. Further, by reducing the distance between the tip of the capillary 102 and the like and the electric stage 3, the mixed droplet forming unit 110 can be downsized.

  Here, when forming the droplet, it is preferable to apply a voltage larger than the pulse voltage applied between the raw material liquid 8 a and the electric stage 3 between the external electrode 320 and the electric stage 3. In this case, since the electrostatic induction charge 321 appearing at the tip of the external electrode 320 biases the charge distribution of the electrostatic induction charge 361 on the surface of the raw material liquid so as to be the highest at the nozzle center portion, That is, a large electrostatic force acts between the central portion of the raw material liquid surface and the electric stage 3. For this reason, the position where the mixed droplet M is formed can be made more stable. In addition, the external electrode 320 is preferably provided in all of the capillaries 101, 102, and 103, but may be provided in any one of them.

  In addition, this invention is not limited to the said embodiment, It can take a various deformation | transformation aspect. For example, in the above-described embodiment, the degree of symmetry Δ is acquired as information regarding the symmetry of light intensity, but the present invention is not limited to this.

  Needless to say, the external electrode 320 and the control device 12 according to the third embodiment may be applied to the second embodiment.

It is a schematic block diagram which shows the evaluation apparatus of the uniformity of the composition which concerns on 1st Embodiment. FIG. 2 is an enlarged view of the capillary and the substrate 2 in FIG. 1. It is a block diagram which shows the image processing apparatus of FIG. 4A is a conceptual diagram showing coordinates applied to the mixed droplets in the symmetry acquisition unit, and FIG. 4B shows a light intensity profile along the r-axis in FIG. 4A. It is a conceptual diagram. It is a flowchart in the case of evaluating the uniformity of the composition according to the first embodiment. 6A shows the transmitted light image data of the mixed droplet MA, FIG. 6B shows the fluorescence image data derived from the fluorescein of the mixed droplet MA, and FIG. 6C shows the rhodamine of the mixed droplet MA. Fluorescence image data derived from B, FIG. 6 (d) is a diagram showing a fluorescence intensity profile along the horizontal line of FIG. 6 (b), and FIG. 6 (e) is along the horizontal line of FIG. 6 (c). It is a figure which shows the profile of fluorescence intensity. FIG. 7A shows the transmitted light image data of the mixed droplet MB, FIG. 7B shows the fluorescence image data derived from the fluorescein of the mixed droplet MB, and FIG. 7C shows the rhodamine of the mixed droplet MB. Fluorescence image data derived from B, FIG. 7D is a diagram showing a fluorescence intensity profile along the horizontal line in FIG. 7B, and FIG. 7E is along the horizontal line in FIG. 7C. It is a figure which shows the profile of fluorescence intensity. It is a schematic block diagram which shows the evaluation apparatus of the uniformity of the composition which concerns on 2nd Embodiment. It is a block diagram which shows the image processing apparatus of FIG. It is a flowchart in the case of evaluating the uniformity of the composition concerning a 2nd embodiment. It is an enlarged view of the capillary and the board | substrate 2 in the evaluation apparatus of the uniformity of the composition which concerns on 3rd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 2 ... Board | substrate, 9 ... Camera, 104 ... Electrode, 320 ... External electrode, 12 ... Control apparatus, 58 ... Center acquisition part (center acquisition means), 60 ... Symmetry acquisition part (symmetry acquisition means), R ... Raw material liquid Drop jet flow, M ... Mixed droplet, 100,200 ... Evaluation device for composition uniformity, 105 ... Raw material injection unit, 107 ... Voltage application device, 115 ... Image acquisition means.

Claims (11)

In the method for evaluating the uniformity of the composition in the mixed droplet formed by mixing a plurality of raw material liquids having different compositions from each other,
An image acquisition step of collecting and imaging light that reaches from the mixed droplet with an intensity corresponding to the amount of any component of the mixed droplet, and acquiring image information about the mixed droplet;
A center acquisition step of acquiring a center position of the mixed droplet in the image information;
In the image information, a symmetry acquisition step of acquiring information on the symmetry of the light intensity with respect to the center position of the mixed droplet;
The evaluation method of the uniformity of the composition containing this. The raw material liquid includes a component that absorbs light of a predetermined wavelength,
In the image acquisition step, the mixed droplets are irradiated with light of the predetermined wavelength, and the transmitted light that reaches through the mixed droplets is collected and imaged to acquire the image information. The method for evaluating the uniformity of the composition according to claim 1. The raw material liquid includes a fluorescent dye that emits fluorescence when irradiated with excitation light,
2. The image acquisition step acquires the image information by irradiating the mixed droplet with the excitation light and condensing and imaging the fluorescence generated and reached by the excitation light irradiation. Evaluation method of uniformity of composition. Further, the step of irradiating the mixed droplets with light, condensing the transmitted light transmitted through the mixed droplets to form an image, and acquiring transmitted light image information about the mixed droplets,
The composition uniformity evaluation method according to claim 3, further comprising a correction step of correcting the image information based on the transmitted light image information before performing the symmetry acquisition step. The center acquisition step acquires the outer edge shape of the mixed droplet in the image information, approximates the outer edge shape to an ellipse including a perfect circle, and sets the center position of the ellipse including the perfect circle as the center of the mixed droplet The uniformity evaluation method according to any one of claims 1 to 4, which is acquired as a position. The center acquisition step acquires an area occupied by the mixed droplet in the image information, and acquires a center of gravity of the mixed droplet area as a center position of the mixed droplet. The evaluation method of the uniformity of the composition as described in 1. 5. The center acquisition step acquires the center of gravity of the light intensity distribution in the image information, and acquires the position of the center of gravity of the light intensity distribution as the center position of the mixed droplet. The evaluation method of the uniformity of the composition as described in 1. The mixed droplets apply a pulse voltage between the raw material liquid stored for each of the plurality of capillaries and a substrate disposed opposite to the tip of the capillary, and the raw material liquid is discharged from the tip of each capillary. The method for evaluating the uniformity of the composition according to claim 1, wherein the composition is formed on the substrate by being injected. The light that reaches from the mixed droplets formed by mixing a plurality of raw material liquids having different compositions to each other with the intensity corresponding to the amount of any component in the mixed droplets is condensed to form an image. Image information acquisition means for acquiring image information about the droplet;
Center acquisition means for acquiring a center position of the mixed droplet in the image information;
In the image information, symmetry acquisition means for acquiring information on symmetry of the intensity of the light with respect to the center position of the mixed droplet;
An apparatus for evaluating the uniformity of a composition. A raw material liquid injection section having a plurality of capillaries each storing the raw material liquid for forming the mixed droplet;
A substrate on which the mixed liquid droplets formed from the raw material liquid, which is arranged facing the tip of the capillary and ejected from the tip of each capillary, is placed;
A voltage applying device that applies a pulse voltage between each of the raw material liquids stored in the capillaries and the substrate;
The apparatus for evaluating uniformity of composition according to claim 9, further comprising: Wherein among the plurality of capillaries are installed electrodes on the outer periphery of the at least one capillary,
The composition uniformity evaluation device according to claim 10, further comprising a control device that controls the voltage application device so that a potential equal to or higher than the potential of the raw material liquid is applied to the electrode . evaluation device.

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