JP5338439B2 - Microinjection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enlarge the tolerance of injection calibration by considering dispersion of inner diameters of capillary needles in the production, and combination of introducing materials. <P>SOLUTION: The microinjection device 100 is constituted so that an image including a brightness value within a prescribed range can be acquired and the brightness value used for the injection calibration can be acquired accurately by taking the image of an objective substance containing a fluorescent reagent mixed therewith by a prescribed exposure time, acquiring the maximum brightness value from the taken image, determining the exposure time by which the brightness value is included in the prescribed range when the maximum brightness value is not included in the prescribed range, and regulating the exposure time to the determined exposure time. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a microinjection apparatus. For example, the present invention relates to a microinjection apparatus that expands a calibration allowable range of discharge calibration caused by a combination of a capillary needle and an introduced substance to an extent that causes no practical problem by adjusting an exposure time.

  In recent years, microinjection devices have been used when biomolecules such as genes and proteins and compounds (hereinafter simply referred to as introduction substances) are injected into cells. The microinjection device has the advantage that the combination of the introduced substance and the introduced cell is free.

In view of the advantages of this microinjection device, for example, creation of artificial pluripotent stem cells (IPS cells) using genes as introduction substances, functional analysis using proteins as introduction substances, drug development using compounds as introduction substances, etc. Application to is expected.

  The microinjection device has a fine hollow glass needle (hereinafter referred to as a capillary needle), and after the introduction substance is filled into the capillary needle, the cell is pierced and discharged into the cell. Inject into cells.

  The microinjection apparatus is required to quantitatively discharge the introduced substance from the above-mentioned purpose of use, and the following techniques are known for this demand.

  First, as a technique for quantitatively controlling the amount of microinjection dispensed into cells in subpicoliter units, a regulated air pressure is applied to a capillary needle filled with the introduced substance, and a predetermined amount of introduced substance is discharged into the cell. The technique of doing is known (for example, refer patent document 1).

  Further, a method is known in which a predetermined amount of an introduced substance is discharged into a cell using a pressure for discharging the introduced substance into the cell and a pressure for preventing the introduced substance from flowing back into the capillary needle (for example, , See Patent Document 2).

  Furthermore, the fluorescent reagent is mixed with the introduction substance, the volume for discharging the introduction substance is adjusted based on the luminance value data indicated by the fluorescence reagent, and quantitative discharge is realized by discharging the adjusted volume. A technique is known (see Patent Document 3). This technique will be described in detail below.

In this case, the volume V into which the introduced substance is discharged is as follows, where the capillary needle tip inner diameter is D, the viscosity of the introduced substance is μ, the discharge pressure applied to the capillary needle is Pi, and the discharge pressure duration is Ti. It is represented by the formula (1) shown.

From the equation (1), the volume V is proportional to the product of the discharge pressure and the discharge time (hereinafter referred to as discharge parameter), using the capillary needle tip inner diameter to be used and the viscosity of the introduced substance as parameters. Is represented by equation (2). Note that η is a discharge coefficient.

  Therefore, if the discharge coefficient η is known, the volume V corresponding to each discharge parameter can be obtained, and the introduced substance can be discharged quantitatively. Hereinafter, a process of determining η in the above-described formula (2) using a fluorescent reagent, that is, “discharge calibration” will be described.

  As a preliminary step of discharge calibration, a relationship between the volume V of the fluorescent reagent and the fluorescence intensity I indicated by the fluorescent reagent is determined using a microinjection apparatus 50 shown in FIG. However, in the pre-discharge calibration step, the petri dish is filled with paraffin oil or silicon oil having a small surface tension instead of the medium. After describing the microinjection apparatus 50, the relationship between the volume V and the fluorescence intensity I will be described.

  The unit of the volume V is expressed by “pl: picoliter”, and the unit of the fluorescence intensity I defined as the sum of the luminance values of all the pixels of the fluorescence image will be described below as the pixel luminance value.

  FIG. 19 is a diagram for explaining a preliminary step of discharge calibration performed using a conventional microinjection apparatus. The microinjection apparatus 50 shown in FIG. 19 discharges the fluorescent reagent from the capillary needle 50a into paraffin oil. The fluorescent reagent discharged into the paraffin oil forms a spherical fluorescent droplet 50b because of the surface tension.

  Next, light having a wavelength other than the wavelength emitted from the fluorescent reagent is removed by the fluorescent filter 50f, a fluorescent image of the fluorescent droplet 50b is acquired by the high-sensitivity CCD camera 50g, and the diameter of the droplet is measured. The volume V of the fluorescent droplet 50b is obtained.

  And the fluorescence intensity I is calculated | required from the above-mentioned fluorescence image, and correlation with the volume V of a fluorescence reagent is calculated | required from the graph 5 shown below in FIG. FIG. 20 is a diagram showing the relationship between fluorescence intensity and volume.

From the graph 5 in which the X axis shown in FIG. 20 is the fluorescence intensity I and the Y axis is the volume V, the proportionality constant α (reagent constant) in the equation (3) of the fluorescence intensity I and the volume V is obtained. Pre-steps for completing are complete. In addition, what is necessary is just to implement this step once for every fluorescence reagent.

  Next, the discharge calibration performed with the combination of the capillary needle to be used and the introduced substance immediately before the injection work for introducing the desired substance into the cell will be described. FIG. 21 is a diagram for explaining the discharge calibration performed for each combination of the capillary needle and the introduced substance.

  As shown in FIG. 21, the fluorescent reagent 50h is discharged a plurality of times by changing the value of the discharge parameter from the capillary needle 50a.

  Thus, when performing discharge calibration, discharging the introduced substance a plurality of times is referred to as “test discharge”. At the time of this test discharge, fluorescent reagent data relating to the fluorescent reagent 50h is input.

  The fluorescent reagent data includes the reagent constant α obtained in the previous step, the concentration ρ0 of the fluorescent reagent when the reagent constant is obtained, the exposure time Te0 of the high sensitivity CCD camera 50g, and the like. Note that ρ0 is a reference density and Te0 is a reference exposure time.

  Next, measurement of the fluorescence intensity indicated by the fluorescent reagent 50h discharged in FIG. 21 will be described. This fluorescence intensity is measured based on a fluorescence image acquired for each discharge. This will be specifically described below with reference to the drawings.

  FIG. 22 is a diagram for explaining acquisition of fluorescence intensity. The background image 20 shown in FIG. 22 shows an image before the fluorescent reagent 50h is discharged from the capillary needle 50a, and the fluorescent image 21 is acquired when the fluorescent reagent 50h is discharged from the capillary needle 50a.

And the difference image 22 is acquired from the background image 20 and the fluorescence image 21, and the fluorescence intensity I is _{calculated |} required from the sum total of the pixel brightness _| luminance _Jxy in the acquired difference image 22. FIG. The formula for calculating the fluorescence intensity I is defined as the following formula (4).

  Then, the relationship between the discharge parameter and the fluorescence intensity is obtained using the fluorescence intensity I obtained from the equation (4). FIG. 23 is a diagram for explaining the relationship between ejection parameters and fluorescence intensity. In the graph of FIG. 23, the X axis is the discharge parameter Pi · Ti, and the Y axis is the fluorescence intensity I.

From the graph 11 shown in FIG. 23, the relationship between the fluorescence intensity I and the ejection parameter Pi · Ti is expressed by the equation (5), and the proportionality constant γ is obtained.

  Then, by combining Expression (3) and Expression (5), the discharge coefficient η = αγ of Expression (2) described above is derived, and the volume V with respect to the discharge parameter is calculated. The relationship between the discharge parameter and the volume V will be described. FIG. 24 is a diagram showing the discharge calibration result.

  From the graph 12 with the discharge parameter Pi · Ti shown in FIG. 24 as the X axis and the volume V as the Y axis, the volume V is calculated for each arbitrary discharge parameter, and calibrated discharge becomes possible. As a result, the volume V discharged from the capillary needle 50a can be set arbitrarily.

  The relationship between the reference density ρ0 and the reference exposure time Te0 and the formula (2) is shown. In the case of an arbitrary density ρ and an arbitrary exposure time Te, ρ0 / ρ and the volume indicated by each value indicated by Te0 / Te. Since V is generally proportional, these may be calculated by multiplying the right side.

  Next, the flow of discharge calibration that has been described so far will be described. FIG. 25 is a flowchart showing conventional discharge calibration. First, the concentration ρ of the fluorescent reagent that is actually used is input to the microinjection apparatus 50 (step S50).

  Next, the microinjection apparatus 50 reads the fluorescent reagent data obtained and stored in advance (step S51). Then, the exposure time is set to a predetermined Te (step S52).

  Then, a fluorescent image for discharge calibration is acquired (step S53), and a volume V corresponding to each discharge parameter is obtained from the acquired fluorescent image (step S54). And the result of the discharge calibration which makes the graph 12 shown in FIG. 24 an example is acquired (step S55).

  Next, the fluorescence image acquired in step S53 described above and the luminance profile included in the acquired fluorescence image will be described. FIG. 26 is a diagram for explaining the fluorescence image and the luminance profile.

  The fluorescent image 23 shown in FIG. 26 shows an example of a fluorescent image obtained when the discharge volume V differs depending on the combination of the capillary needle 50a and the introduced substance, and is taken with a predetermined exposure time Te. And the luminance profile acquired from the fluorescence image 23 becomes the luminance profiles 30 to 32 shown in the figure.

  The luminance profile is the luminance distribution in the X direction of the fluorescent image (Y coordinate is the center of the screen). In the example shown in FIG. 26, the luminance profile 30 has the highest pixel luminance, and the pixel luminances in the order of the luminance profiles 31 and 32. The maximum value of becomes smaller.

  The maximum pixel brightness indicated by the brightness profiles 30 to 32 shown in FIG. 26 is a value equal to or lower than the upper limit Js shown by the high-sensitivity CCD camera 50g shown in FIG. Based on this, discharge calibration is performed.

  The upper limit Js indicates the maximum pixel luminance that can be captured by the high-sensitivity CCD camera 50g. The pixel luminance exceeding the upper limit Js is a saturated region, and the maximum pixel luminance indicated in the saturated region cannot be measured. In this case, it is difficult to obtain the fluorescence intensity necessary for discharge calibration.

  Note that, as shown in the luminance profiles 30 to 32 shown in FIG. 26, the luminance distribution of the fluorescent image is basically a substantially two-dimensional normal distribution due to the diffusion of the fluorescent reagent into the medium.

Japanese Patent Laid-Open No. 2007-300868 JP 2008-86242 A JP 2007-289081 A

  However, the above-described conventional technique has a problem that the allowable range of discharge calibration is narrow and cannot be flexibly calibrated when considering the manufacturing variation of the inner diameter of the capillary needle and the combination of introduced substances.

  This will be specifically described with reference to the drawings. FIG. 27 is a diagram for explaining the problem. In the luminance profile 40 shown in FIG. 27, the discharge volume V is extremely small, the SN ratio between the pixel luminance and the background noise is poor, and the measurement error increases when the fluorescence intensity I is measured.

  The luminance profile 41 is shown in a saturation region where the discharge volume V is extremely large and the maximum value of the true luminance profile exceeds the upper limit Js. In this case, the fluorescence intensity I cannot be measured correctly, and accurate discharge calibration becomes difficult as in the case of the luminance profile 40.

  As described above, it is difficult to perform accurate discharge calibration unless the maximum value indicated by the luminance profile is less than the upper limit Js and within a range not affected by the background noise.

  The disclosed technique has been made in view of the above, and provides a microinjection apparatus that expands the allowable calibration range of discharge calibration caused by a combination of a capillary needle and an introduced substance to a level that does not cause a problem in practice. Objective.

  The microinjection device disclosed in the present application includes a fluorescent image acquisition unit that mixes a fluorescent reagent into a target object to be injected into a cell, captures the fluorescent reagent with a predetermined exposure time, and acquires a fluorescent image; A determination unit that acquires a luminance value of the maximum pixel luminance from the image and determines whether or not the luminance value is included in the predetermined range; and the predetermined value when the luminance value is not included in the predetermined range. It is a requirement to have an exposure time adjustment unit that determines an exposure time that includes a luminance value within the range and adjusts the predetermined exposure time according to the determined exposure time.

According to the microinjection apparatus, by adjusting the exposure time, to expand the maximum pixel intensity obtained from the fluorescence images was adjusted within a predetermined _range, the allowable range of discharge calibration extent no practical problem Can do.

FIG. 1 is a diagram for explaining the outline of the first embodiment. FIG. 2 is a diagram illustrating the microinjection apparatus according to the first embodiment. FIG. 3 is a diagram illustrating an example of a constant table. FIG. 4 is a flowchart showing processing of the microinjection apparatus shown in the first embodiment. FIG. 5 is a flowchart illustrating processing of the exposure time control unit according to the first embodiment. FIG. 6 is a diagram for explaining the effect of the first embodiment. FIG. 7 is a diagram for explaining a case where the discharge volume is extremely large. FIG. 8 is a diagram illustrating the microinjection apparatus according to the second embodiment. FIG. 9 is a diagram for explaining a luminance distribution that forms a two-dimensional normal distribution with a fluorescent image. FIG. 10 is a diagram for explaining the relationship between the saturated pixel number ratio and the saturated pixel luminance. FIG. 11 is a diagram illustrating the relationship between the saturated pixel number ratio and the saturated pixel luminance. FIG. 12 is a flowchart illustrating the process of the microinjection apparatus according to the second embodiment. FIG. 13 is a diagram illustrating a microinjection apparatus according to a third embodiment. FIG. 14 is a diagram for explaining the relationship between the threshold pixel number ratio and the luminance distribution. FIG. 15 is a diagram for explaining saturated pixel luminance when σ is arbitrarily set. FIG. 16 is a flowchart illustrating the process of the microinjection device according to the third embodiment. FIG. 17 is a diagram illustrating the microinjection apparatus according to the fourth embodiment. FIG. 18 is a flowchart illustrating the process of the microinjection device according to the fourth embodiment. FIG. 19 is a diagram for explaining a preliminary step of discharge calibration performed using a conventional microinjection apparatus. FIG. 20 is a diagram showing the relationship between fluorescence intensity and volume. FIG. 21 is a diagram for explaining the discharge calibration performed for each combination of the capillary needle and the introduced substance. FIG. 22 is a diagram for explaining acquisition of fluorescence intensity. FIG. 23 is a diagram for explaining the relationship between ejection parameters and fluorescence intensity. FIG. 24 is a diagram for explaining the discharge calibration result. FIG. 25 is a flowchart for explaining conventional discharge calibration. FIG. 26 is a diagram for explaining the fluorescence image and the luminance profile. FIG. 27 is a diagram for explaining the problem.

  Hereinafter, embodiments of a microinjection apparatus disclosed in the present application will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  First, an outline of the microinjection apparatus shown in the first embodiment will be described. The microinjection apparatus shown in the first embodiment adjusts the exposure time during discharge calibration.

By adjusting the exposure time, the maximum pixel luminance value of the luminance profile acquired from the fluorescent image is not affected by background noise from a predetermined value that does not exceed the upper limit Js (hereinafter referred to as J _HIGH ). The range is adjusted to a predetermined value (hereinafter referred to as J _LOW ).

This will be specifically described with reference to the drawings. FIG. 1 is a diagram for explaining the outline of the first embodiment. Microinjection apparatus 100 shown in FIG. 1, when the maximum pixel intensity value of the luminance profile 90 obtained from the fluorescence image (simply, a J _MAX) is saturated to limit Js the acquired J _MAX The exposure time is adjusted so that the adjusted J _A is acquired from the fluorescent image by adjusting the value to be lower than the upper limit Js (for example, J _A ).

_{Meanwhile, J MAX} is, if a value affected by background _noise, for adjusting the _{J MAX} to have a value within the range shown a _{J MAX J HIGH} and _{J LOW} is. Then, the exposure time is adjusted so that the adjusted J _MAX (for example, “J _A ”) is acquired from the fluorescent image.

In this way, the microinjection apparatus adjusts the exposure time, thereby adjusting the maximum pixel luminance indicated by the luminance profile to a value within the range defined by J _HIGH and J _Low , thereby increasing the discharge calibration allowable range. It can be expanded to the extent that there is no practical problem.

In the following a description will be given of an exposure time maximum pixel intensity J _A shown in FIG. 1 is obtained as "optimum exposure time".

  Next, the microinjection apparatus illustrated in FIG. 1 will be described. FIG. 2 is a diagram illustrating the microinjection apparatus according to the first embodiment. The microinjection apparatus 100 shown in FIG. 2 accurately acquires exposure calibration data by appropriately adjusting the exposure time during discharge calibration, and discharges the introduced substance into the cell based on the acquired data. .

  The microinjection apparatus 100 includes a control unit 101, a capillary needle operation unit 102, a capillary needle 103, a petri dish 104, a microscope 105, a high-sensitivity CCD camera 106, a fluorescent filter 107, a reflecting mirror 108, An excitation filter 109 and an excitation light source 110 are included.

  The control unit 101 calculates the discharge parameter Pi · Ti from the desired discharge volume V based on the equation (2) described in the prior art, and the capillary needle operation unit discharges the introduction material of the desired volume V. A processing unit for instructing 102.

Furthermore, as a function closely related to the present embodiment, the control unit 101 acquires a fluorescence image when performing discharge calibration on the introduced introduction substance and the capillary needle 103. Then, the maximum pixel brightness J _MAX is calculated from the acquired fluorescence image, and the exposure time is adjusted based on the calculated J _MAX .

  The control unit 101 includes a storage unit 101a, an ejection control unit 101b, an image acquisition unit 101c, and an exposure time control unit 101d.

The storage unit 101a stores unique data (fluorescent reagent type, reagent constant α, reference concentration ρ0, reference exposure time Te0), data combining the discharge pressure Pi and the discharge pressure duration Ti, and the maximum discharge pressure. The value Pi _MAX has a maximum value Ti _MAX of the discharge pressure duration.

  Further, the storage unit 101a has a constant table 111 in addition to the various data described above. FIG. 3 is a diagram illustrating an example of the data structure of the constant table.

The constant table 111 shown in FIG. 3 stores the upper limit J _HIGH and the lower limit J _LOW of the adjustment target. Further, the upper limit Te _HIGH and Te _{LOW of} the exposure time are stored.

Further, as described above, J _HIGH is a predetermined value that does not exceed the upper limit Js, and J _LOW is a predetermined value that is not affected by the background noise.

  Returning to the description of FIG. 2, the discharge controller 101b will be described. The ejection control unit 101b is a processing unit that controls the volume to be ejected based on various data stored in the storage unit 101a.

At the time of discharge calibration, the discharge controller 101b sets the exposure time Te of the high-sensitivity CCD camera 106 to the initial exposure time Te1, acquires the discharge pressure Pi _MAX and the discharge time Ti _MAX from the storage unit 101a, and acquires the acquired Pi. _A discharge command is output by _MAX and Ti _MAX . The product of the discharge pressure and the discharge time will be described below as a discharge parameter.

  The image acquisition unit 101c is a unit that acquires a fluorescent image during discharge calibration and outputs the acquired image to the exposure time control unit 101d. This fluorescent image is an image taken by the high sensitivity CCD camera 106. As an example, there is a fluorescent image 23 shown in FIG.

The exposure time control unit 101d calculates the maximum pixel brightness J _MAX from the fluorescence image acquired from the image acquisition unit 101c, and when the calculated J _MAX is in the range of J _HIGH and J _LOW , the J _MAX is captured. The time is determined as the optimum exposure time TeB.

Specifically, a description will be given using the constant table 111 shown in FIG. First, the exposure time control unit 101d acquires a fluorescent image from the image acquisition unit 101c. Then, J _MAX is calculated from the acquired fluorescent image using a conventional technique.

Then, the exposure time control unit 101d acquires J _HIGH and J _LOW stored in the constant table 111 and compares them with the calculated J _MAX . When the exposure time control unit 101d determines that J _MAX does not exceed J _HIGH (J _MAX <J _HIGH ), the exposure time control unit 101d compares the values of J _MAX and J _LOW .

In this case, the exposure time control unit 101d, determined by comparing the obtained _{J MAX} and _{J LOW,} if _{J MAX} is not less than _{J LOW (J MAX> J LOW} ), the initial exposure time Te1 as TeB optimum exposure time To do.

On the other hand, the exposure time control unit 101d, the results of comparison of _{J MAX} and the calculated _{J HIGH} obtained from constant table _{111, if J MAX} is determined to be beyond the _{J HIGH (J MAX> J HIGH} ), the initial The exposure time Te1 is multiplied by a fixed magnification (eg, 0.65 times), and the multiplied value Te2 is output to the ejection control unit 101b.

Thereafter, the ejection control unit 101b determines whether Te2 input by the exposure time control unit 101d is less than the lower limit Te _{LOW of the} exposure time. When Te2 is not less than Te _LOW (Te2> Te _LOW ), the ejection control unit 101b updates the exposure time Te to Te2 and issues a ejection command to the capillary needle operation unit 102 again.

On the other hand, when Te2 is less than Te _LOW (Te2 <Te _LOW ), the discharge controller 101b does not update the initial exposure time Te1 to Te2, and suspends discharge calibration on the assumption that the discharge volume exceeds the calibration allowable range. .

Further, the exposure time control unit 101d compares the acquired J _MAX and J _LOW , and when J _MAX is less than J _LOW (J _MAX <J _LOW ), C1 = (J _HIGH + J _LOW ) / 2 and the ratio of J _MAX (C1 / J _MAX ), and outputs the multiplied value Te2 to the discharge control unit 101b.

For example, when J _MAX = “5”, J _LOW = “10”, and J _HIGH = “20”, C1 is “15”, and the value obtained by multiplying the initial exposure time Te1 by three from the ratio with J _MAX is Te2. It becomes. In this case, when the image is taken at the exposure time Te2, J _MAX is “15” and has a value in the range of J _HIGH and J _LOW .

Then, the ejection control unit 101b determines whether or not Te2 input by the exposure time control unit 101d exceeds the upper limit Te _{HIGH of the} exposure time. When Te2 exceeds Te _HIGH (Te2> Te _HIGH ), the discharge controller 101b does not update the initial exposure time Te1 to Te2, and suspends discharge calibration assuming that the discharge volume V is below the allowable calibration range. .

On the other hand, when Te2 input by the exposure time control unit 101d is less than Te _HIGH (Te2 <Te _HIGH ), the discharge control unit 101b updates the exposure time Te to Te2 and sends a discharge command to the capillary needle operation unit 102. Again.

Thus, when the calculated J _MAX exceeds J _HIGH , the exposure time control unit 101d multiplies the exposure time corresponding to J _MAX by 0.65, and when it is less than J _LOW , J J multiplying _{C1 =} exposure time corresponding to _{MAX (J HIGH + J LOW)} / 2 and _{J MAX} ratio of (C1 _{/ J MAX).}

If the calculated J _MAX is less than J _HIGH and exceeds J _LOW , the above-described processing is not performed, and ejection is performed at the exposure time Te1. In this way, the exposure time for taking a fluorescent image that can be acquired by J _MAX that is less than J _HIGH and exceeds J _LOW will be described below as the optimum exposure time TeB.

  The capillary needle operation unit 102 receives the discharge calibration command from the control unit 101 during discharge calibration, and discharges the introduced material. After discharge calibration, the capillary needle operation unit 102 discharges the introduced material based on data output from the control unit 101.

  The capillary needle 103 is attached to the capillary needle operation unit 102 and discharges the introduced substance filled therein based on data output from the control unit 101.

  The petri dish 104 shows a container that fills the medium 50d shown in FIG. 21, and the microscope 105 is an apparatus that observes the introduced substance that diffuses on the petri dish.

  The high-sensitivity CCD camera 106 is a device that converts an optical image into electronic image data using a video device, and takes a fluorescent image. Further, the exposure time at the time of shooting is controlled by the control unit 101.

  The fluorescence filter 107 is a filter that removes light having a wavelength other than the fluorescence wavelength from the fluorescence image obtained by the high-sensitivity CCD camera 106 and forms an image on the image element of the fluorescence image.

  The reflecting mirror 108 is a mirror for reflecting the state observed by the microscope 105 toward the high-sensitivity CCD camera 106, and directs the excitation light emitted from the excitation light source 110 toward the introduced substance discharged from the capillary needle 103. Reflect.

  The excitation light source 110 is a light source for irradiating the introduction material with excitation light when the introduction material containing the fluorescent material is ejected from the tip of the capillary needle 103.

  Next, the process of the microinjection apparatus 100 shown in Example 1 will be described. FIG. 4 is a flowchart showing processing of the microinjection apparatus shown in the first embodiment. First, the concentration ρ of the fluorescent reagent actually used is input to the microinjection apparatus 100 (step S90).

  Next, the microinjection apparatus 100 reads the fluorescence reagent data that has been obtained and stored in advance (step S91). Then, the exposure time control unit 101d adjusts the exposure time (step S92).

  Then, after the exposure time adjusted in step S102 is set (step S93), ejection calibration data is acquired (step S94). Thereafter, the volume V is calculated based on the acquired data (step S95). And the result of discharge calibration is acquired (step S96).

  Next, the process of the exposure time adjustment unit 101d shown in the first embodiment will be described. FIG. 5 is a flowchart showing processing of the exposure time control unit shown in the first embodiment.

Data obtained by combining the discharge pressure Pi and the discharge pressure duration Ti, the discharge pressure maximum value Pi _MAX , the discharge pressure duration maximum value Ti _{MAX and the} like are input to the microinjection apparatus 100 (step S100). .

  The various data input in step S100 is stored in the storage unit 101a (step S101). The ejection control unit 101b sets the initial exposure time Te1 of the high sensitivity CCD camera 106 (step S102).

Then, the discharge control unit 101b acquires the discharge pressure Pi _MAX and the discharge time Ti _MAX from the storage unit 101a, and outputs a discharge command based on the acquired Pi _MAX and Ti _MAX (step S103).

Thereafter, the image acquisition unit 101c acquires a fluorescent image (step S104), and the exposure time control unit 101d acquires the maximum pixel luminance J _MAX from the acquired fluorescent image (step S105).

Then, the exposure time control unit 101d compares the J _MAX acquired in step S105 with the J _HIGH stored in the storage unit 101a, and when J _MAX exceeds J _HIGH (step S106, Yes), the step The initial exposure time set in S102 is multiplied by a fixed magnification (for example, 0.65 times) (Step S107).

Then, the ejection control unit 101b determines whether or not the exposure time Te2 calculated in step S107 is less than the lower limit Te _{LOW of the} exposure time. If Te2 is not less than Te _LOW (No in step S108), step S103. Migrate to

On the other hand, if Te2 is less than Te _LOW (step S108, Yes), it is determined that the discharge volume exceeds the allowable calibration range, and the error is interrupted (step S109).

If J _MAX does not exceed J _HIGH from the comparison result shown in step S106 (No in step S106), the process proceeds to step S110 and is stored in the J _MAX acquired in step S105 and the storage unit 101a. Compare J _LOW .

From the comparison result, when J _MAX exceeds J _LOW (No in step S110), the discharge control unit 101b determines the exposure time set in step S102 as the optimum exposure time TeB (step S114).

On the other hand, when J _MAX does not exceed J _LOW (step S110, Yes), the exposure time control unit 101d has a ratio of C1 = (J _HIGH + J _LOW ) / 2 and J _MAX to the exposure time set in step S102. Multiply by (C1 / J _MAX ) (step S111).

Then, the discharge control unit 101b compares the exposure time Te2 calculated in step S111 with the upper limit Te _HIGH of the exposure time. If Te2 does not exceed Te _HIGH (No in step S112), the process proceeds to step S103. Transition.

On the other hand, if Te2 exceeds Te _HIGH (step S112, Yes), an error is interrupted because the discharge volume is below the allowable calibration range (step S113).

According to this flow chart, the exposure time control unit 101d is, by adjusting the exposure time, to adjust the maximum pixel intensity _{J MAX} to the value of the range defined by _{J HIGH} and _{J LOW,} practical tolerance of discharge calibration It can be expanded to the extent that there is no problem.

As described above, according to the microinjection apparatus shown in the first embodiment, the maximum pixel luminance can be adjusted to a value in a range defined by J _HIGH and J _LOW by adjusting the exposure time. This will be specifically described below with reference to the drawings.

FIG. 6 is a diagram for explaining the effect of the first embodiment. The example shown on the left side of FIG. 6 is a case where the maximum value J ₀ of the luminance profile exceeds the upper limit Js, and the maximum pixel luminance J _MAX is equal to the upper limit Js. In this case, the maximum pixel brightness J _MAX is adjusted to a value in the range of J _HIGH and J _{LOW in} the second test ejection.

On the other hand, the example shown on the right side of FIG. 6 is affected by background noise because the maximum pixel luminance J _MAX of the luminance profile is too small. In this case as well, the maximum pixel brightness J _MAX is adjusted to a value in the range of J _HIGH and J _{LOW in} the second test discharge as in the case shown on the left side.

By the way, the microinjection apparatus 100 shown in Embodiment 1 reduces the exposure time by 0.65 times per test discharge when the calculated maximum pixel luminance J _MAX is equal to the upper limit Js.

  Therefore, in the case of a capillary needle whose discharge volume is extremely larger than the average one (for example, 10 times or more), only the exposure time is reduced by 0.65 times in one test discharge.

  In such a case, five or more test ejections are required to adjust the maximum pixel luminance within the adjustment target range. Hereinafter, it demonstrates concretely using figures. FIG. 7 is a diagram for explaining a case where the discharge volume is extremely large.

As shown in FIG. 7, the maximum pixel when the maximum value J ₀ of the first intensity profile 91 is extremely larger than the upper Js, it is 0.65 times in the second discharge calibration, the adjustment target range It has no brightness.

  As a result, five or more test discharges are required to reach the adjustment target range. Considering that a waiting time (about 10 seconds) is usually required for the introduced introduced material to diffuse and distribute almost uniformly throughout the fluorescent image, the test discharge is performed five times in succession. In this case, at least about 40 seconds are required for five test discharges, and the operability and workability are significantly impaired.

Therefore, in the microinjection apparatus 200 shown in the second embodiment, a processing unit that calculates “saturated pixel luminance J ₀ ” shown in FIG. 7 from the fluorescence image is provided in the microinjection apparatus 100 shown in the first embodiment.

The “saturated pixel luminance” described above indicates the true maximum pixel luminance when the maximum value indicated by the luminance profile is included in the saturated region, and this will be described below as “J ₀ ”.

  The microinjection apparatus 200 shown in the second embodiment has substantially the same function as that of the microinjection apparatus 100 shown in FIG. 2, and detailed description of functional units having the same functions as the microinjection apparatus 100 is omitted. .

  FIG. 8 is a diagram illustrating the microinjection apparatus according to the second embodiment. The microinjection apparatus 200 shown in FIG. 8 includes a control unit 101, a capillary needle operation unit 102, a capillary needle 103, a petri dish 104, a microscope 105, a high-sensitivity CCD camera 106, a fluorescent filter 107, and a reflecting mirror. 108, an excitation filter 109, and an excitation light source 110.

  The control unit 101 includes a storage unit 101a, an ejection control unit 101b, an image acquisition unit 101c, an exposure time control unit 101d, and a saturated pixel luminance calculation unit 101e.

  Therefore, the difference from the microinjection apparatus 100 is that the control unit 101 has a function of calculating saturated pixel luminance. Hereinafter, processing performed by the saturated pixel luminance calculation unit 101e will be described.

The saturated pixel luminance calculation unit 101e acquires a ratio (saturated pixel number ratio) of the number of pixels in a saturated region from the fluorescent image to the total pixel number (saturated pixel number ratio), and the saturated pixel luminance J ₀ based on the acquired pixel number ratio. Is a processing unit for calculating.

First, the saturated pixel luminance calculation unit 101e will be described functions to be used for calculating the saturation pixel brightness J _0. In general, the luminance distribution indicated by the fluorescent image is represented by a two-dimensional normal distribution. Therefore, J ₀ is calculated using this function. The luminance distribution indicated by the fluorescent image is simply referred to as “luminance distribution”.

FIG. 9 is a diagram for explaining a luminance distribution that forms a two-dimensional normal distribution with a fluorescent image. FIG. 9 shows a luminance distribution 500 having a two-dimensional normal distribution and a corresponding fluorescent image 24. The horizontal axis of the figure is the xy coordinate, the vertical axis is the pixel luminance, and the luminance distribution is the two-dimensional normal distribution J ₀ exp (−) with the vertical and horizontal sizes of the fluorescent image 24 as “1”. It is shown as (x ² + y ² ) / σ ² ). σ is a standard deviation of distribution, and according to an experiment of the microinjection apparatus 200, σ is about 0.1.

FIG. 10 is a diagram for explaining the relationship between the saturated pixel number ratio and the saturated pixel luminance. In FIG. 10, the two-dimensional normal distribution shown in FIG. 9 is shown in polar coordinate format: J ₀ exp (−r ² / σ ² ).

Here, Js indicates the upper limit, n _s = πr _s ² indicates the ratio of the number of pixels in the saturated region to the number of pixels in the entire image (hereinafter referred to as the saturated pixel number ratio), and J ₀ is as described above. Shows the saturation pixel luminance. Note that the pixel luminance is standardized with the upper limit Js being 1.

  First, the two-dimensional normal distribution 500 shown in FIG. 10 shows the luminance distribution of the fluorescent reagent contained in the introduced substance. The luminance distribution below the upper limit Js (solid line portion in FIG. 10) can be measured, but the luminance distribution exceeding the upper limit Js (dotted line portion in FIG. 9) cannot be measured.

In this case, if the saturated pixel number ratio n _s is calculated from the fluorescent image, the saturated pixel luminance J ₀ can be obtained by the following equation (8).

First, the function indicated by the two-dimensional normal distribution 500 and the radial coordinate for the luminance of the upper Js and r _s, the equation (6) below is obtained.

Since the region where r ≦ r _s is a saturated region, the saturated pixel number ratio n _s is expressed by r _{s as shown} in the following formula (7).

From the equations (6) and (7), the relationship between the saturated pixel number ratio n _s and the saturated pixel luminance J ₀ is obtained, and the following equation (8) is obtained.

For example, when Js = 1 and σ = 0.1, πσ ² ≈0.03, and the value of “C” shown in Expression (8) is C = 0.03. The calculation result in this case is shown in FIG. 11 with the saturated pixel number ratio n _{s on} the X axis and the saturated pixel luminance J ₀ on the Y axis.

From the graph 14 shown in FIG. 11, the logarithm of the value of the saturation pixel intensity J ₀ it is found to be proportional to the saturation pixel ratio n _s.

  Next, based on the relationship shown in FIG. 11, taking a fluorescent image at the initial exposure time Te1, and taking the case where the value of the maximum pixel luminance acquired from the fluorescent image is the upper limit Js as an example, the saturated pixel luminance calculation Processing performed by the unit 101e will be described.

First, the saturated pixel luminance calculation unit 101e acquires a fluorescence image from the image acquisition unit 101c, and calculates the pixel number ratio n _s of the saturation region from the acquired fluorescence image. Then, _the saturated pixel luminance J ₀ is calculated by substituting the calculated _ns into the equation (8).

Then, by multiplying the initial exposure time Te1 by C1 / J ₀ (where C1 = (J _HIGH + J _LOW ) / 2), the saturation pixel luminance J ₀ is set to the center level of the adjustment target range. The exposure time Te2 to be adjusted is calculated and output to the exposure time control unit 101d.

Thereafter, the discharge control unit 101b determines whether or not the acquired exposure time Te2 is less than the lower limit Te _{LOW of the} exposure time. If Te2 is not less than Te _LOW , the initial exposure time Te1 is updated to Te2, and the discharge is performed. Do.

  According to the second embodiment, even when the discharge volume is extremely large, the optimum exposure time TeB can be determined by performing the test discharge two to three times, and the adjustment time of the exposure time that is about two to three times. Shortening is possible.

  Next, processing of the microinjection apparatus 200 shown in the second embodiment will be described. FIG. 12 is a flowchart for explaining processing of the microinjection apparatus 200 shown in the second embodiment.

First, data including the combination of the discharge pressure Pi and the discharge pressure duration Ti, the maximum discharge pressure value Pi _MAX , the maximum discharge pressure duration value Ti _{MAX, and the} like are input to the microinjection apparatus 200 (step). S200).

  Then, the various data input in step S200 is stored in the storage unit 101a (step S201). Thereafter, the ejection control unit 101b sets the initial exposure time Te1 of the high sensitivity CCD camera 106 (step S202).

Then, the discharge control unit 101b acquires the discharge pressure Pi _MAX and the discharge time Ti _MAX from the storage unit 101a, and outputs a discharge command based on the acquired Pi _MAX and Ti _MAX (step S203).

Thereafter, the image acquisition unit 101c acquires a fluorescent image (step S204), and the exposure time control unit 101d acquires the maximum pixel luminance J _MAX from the acquired image (step S205).

Then, the exposure time control unit 101d compares the J _MAX acquired in step S205 with the J _HIGH stored in the storage unit 101a, and if J _MAX exceeds J _HIGH (step S206, Yes), saturation is performed. The pixel luminance calculation unit 101e calculates the saturated pixel number ratio n _s (step S207).

Thereafter, the saturated pixel luminance calculation unit 101e uses the equation (7), and calculates the saturated pixel intensity _{J 0,} and outputs the exposure time control unit 101b (step S208).

The exposure time control unit 101d outputs a value Te2 obtained by multiplying the C1 / _{J 0} to the initial exposure time Te1 the ejection control unit 101b (step S209).

Then, the ejection control unit 101b determines whether or not the exposure time Te2 calculated in step S209 is less than the lower limit Te _{LOW of the} exposure time. If Te2 is not less than Te _LOW (No in step S210), step S203. Migrate to

On the other hand, when Te2 is less than Te _LOW (step S210, Yes), an error is interrupted on the assumption that the discharge volume exceeds the calibration allowable range (step S211).

If J _MAX does not exceed J _HIGH from the comparison result shown in step S206 (step S206, No), the process proceeds to step S212, and J _MAX acquired in step S205 and J stored in the storage unit 101a. Compare _LOW .

From the comparison result, when J _MAX exceeds J _LOW (step S212, No), the discharge calibration determines the exposure time Te2 set in step S202 as the optimum exposure time TeB (step S216).

On the other hand, when J _MAX does not exceed J _LOW (step S212, Yes), the exposure time control unit 101d determines the ratio of C1 = (J _HIGH + J _LOW ) / 2 and J _MAX to the exposure time set in step S202. Multiply by (C1 / J _MAX ) (step S213).

Then, the ejection control unit 101b determines whether or not the exposure time Te2 calculated in step S213 is less than the upper limit Te _{HIGH of the} exposure time, and when Te2 does not exceed Te _HIGH (No in step S214), The process proceeds to step S203.

On the other hand, when Te2 exceeds Te _HIGH (step S214, Yes), an error is interrupted on the assumption that the discharge volume is below the allowable calibration range (step S215).

According to this flowchart, the saturated pixel luminance calculator 101e is, by calculating the saturated pixel intensity J _0, even if the discharge volume is extremely large, by adjusting the exposure time of the minimum number of times, the maximum pixel intensity J _MAX was adjusted to a value in the range defined by J _HIGH and J _LOW, it is possible to expand the allowable range of the discharge calibration extent no practical problem.

By the way, the microinjection apparatus 200 shown in Example 2 uses a constant value (for example, 0.1) obtained by investigating the standard deviation σ of the luminance distribution of the fluorescent image in advance, but the introduced substance to be mixed with the fluorescent reagent some types may the value of σ is different, in this case, the calculation accuracy of the saturation pixel intensity J ₀ saturation pixel luminance calculator 101e calculates decreases.

In a third embodiment described below, in addition to the number of saturated pixels ratio n _s, the threshold value Jt provided at a level lower the luminance by a certain percentage from the upper limit Js, ratio entire image pixel brightness above this threshold Jt A means for calculating n _t is provided. Note that n _t is the threshold pixel number ratio.

Then, the microinjection apparatus 300 according to the third embodiment calculates the saturated pixel number ratio n _s and the threshold pixel number ratio n _t described above from the fluorescent image, and calculates the saturated pixel luminance J ₀ from the calculated n _s and n _t. Assume that the processing unit for calculation is provided in the microinjection apparatus 200 shown in the second embodiment.

As in Example 2, the "saturated pixel intensity J _0", the maximum value of the luminance profile, and shows the true maximum pixel brightness when contained in the saturation region, which "J ₀ Will be described below.

  The microinjection apparatus 300 shown in the third embodiment has substantially the same function as the microinjection apparatus 100 shown in FIG. 2, and detailed description of functional units having the same functions as the microinjection apparatus 100 is omitted. .

  FIG. 13 is a diagram illustrating a microinjection apparatus according to a third embodiment. A microinjection apparatus 300 shown in FIG. 13 includes a control unit 101, a capillary needle operation unit 102, a capillary needle 103, a petri dish 104, a microscope 105, a high-sensitivity CCD camera 106, a fluorescent filter 107, and a reflecting mirror. 108, an excitation filter 109, and an excitation light source 110.

  The control unit 101 includes a storage unit 101a, an ejection control unit 101b, an image acquisition unit 101c, an exposure time control unit 101d, a saturated pixel luminance calculation unit 101e, and a threshold pixel number ratio calculation unit 101f.

  Therefore, the difference from the microinjection apparatus 100 is that the control unit 101 includes a saturated pixel luminance calculation unit 101e and a threshold pixel number ratio calculation unit 101f. Hereinafter, processing performed by the saturated pixel luminance calculation unit 101e and the threshold pixel number ratio calculation unit 101f will be described.

The threshold pixel number ratio calculation unit 101f calculates the pixel number ratio n _t of the luminance exceeding the above-described threshold Jt from the fluorescent image, and outputs it to the saturated pixel luminance calculation unit 101e. The saturated luminance calculation unit is a processing unit that calculates a saturated pixel number ratio n _s from the fluorescent image, and calculates a saturated pixel luminance J ₀ from the saturated pixel number ratio n _s and the threshold pixel number ratio n _t .

First, the relationship between the above-described Jt and the pixel number ratio n _t will be described, and then the processing performed by the threshold pixel number ratio calculation unit 101f and the saturated pixel luminance calculation unit 101e will be described.

FIG. 14 is a diagram for explaining the relationship between the threshold pixel number ratio and the luminance distribution. Here, as in FIG. 9, a luminance distribution 600 forming a two-dimensional normal distribution is shown in a polar coordinate format. In the figure, Js represents the upper limit of the imaging system, n _s represents the saturated pixel number ratio, and J ₀ represents the saturated pixel luminance J ₀ as described above. Note that the pixel luminance is standardized with the upper limit Js being 1.

Js · Jt indicates a threshold was lowered pixel luminance by a predetermined ratio (Jt) from the upper limit Js, n _t is the ratio of the number of pixels in the region exceeding js · Jt to the number of pixels of the entire image (hereinafter, a threshold pixel number ratio ).

  First, the two-dimensional normal distribution 600 shown in FIG. 14 shows the luminance distribution of the fluorescent reagent contained in the introduced substance. And although it can measure about luminance distribution (solid line part of Drawing 14) below upper limit Js, it cannot measure about luminance distribution (dotted line part of Drawing 14) exceeding upper limit Js.

In this case, if the saturated pixel number ratio n _s and the threshold pixel number ratio n _t are obtained from the fluorescence image, the saturated pixel luminance J ₀ can be obtained by the following equation (9) even if the standard deviation σ of the distribution is unknown. .

The relationship between the saturated pixel number ratio n _s and the saturated pixel luminance J ₀ is given by Expression (8), as in the second embodiment.

Next, when the ratio (threshold pixel number ratio n _t ) of the number of pixels in the region where the luminance exceeds the threshold value Jt · Js to the total number of pixels from the function indicated by the two-dimensional normal distribution 600 is obtained in a similar manner, The following equation (9) is obtained.

Then, when σ is eliminated from Equation (8) and Equation (9), Equation (10) in which J ₀ is finally calculated from n _s , n _t , Jt, and Js is obtained.

  A case where Jt = 0.5 will be described as an example. FIG. 15 is a diagram for explaining saturated pixel luminance when σ is arbitrarily set.

In FIG. 15, the X axis is the saturated pixel number ratio n _s , and the Y axis is the saturated pixel luminance J ₀ calculated by Expression (10). From these graphs 15 to 17, it is shown that the saturated pixel luminance J ₀ is obtained for an arbitrary σ.

  Next, processing performed by the saturated pixel luminance calculation unit 101e and the threshold pixel number ratio calculation unit 101f will be described based on the above-described calculation formula. The case where the initial exposure time is Te1 and a fluorescent image is taken and the maximum pixel luminance value acquired from the fluorescent image is the upper limit Js will be described as an example. In addition to the various data shown in the first embodiment, Jt: 0.5 is stored in the storage unit 101a.

First, the threshold pixel number ratio calculation unit 101f acquires a fluorescence image from the image acquisition unit 101c, calculates a threshold pixel number ratio n _t from the acquired fluorescence image, and outputs the threshold pixel number ratio n _t to the saturated pixel luminance calculation unit 101e. Next, the saturated pixel luminance calculation unit 101e calculates the saturated pixel number ratio n _s from the same fluorescent image, and obtains the saturated pixel luminance J ₀ from Equation (10) using n _s and n _t .

Then, the initial exposure time Te1 is multiplied by C1 / J ₀ (here, C1 = (J _HIGH + J _LOW ) / 2). Obtains the exposure time Te2 for adjusting the saturation pixel intensity J ₀ in the middle level of the adjustment target range.

Thereafter, it is determined whether or not the exposure time Te2 acquired by the discharge control unit 101b is less than the lower limit Te _{LOW of the} exposure time. If Te2 is not less than Te _LOW , the initial exposure time Te1 is updated to Te2, and the discharge is performed again. I do.

According to this embodiment, the saturated pixel luminance J ₀ can be calculated even when the standard deviation σ of the luminance distribution is unknown by obtaining the values of the saturated pixel number ratio n _s and the threshold pixel number ratio n _t. It becomes.

In the second embodiment and the third embodiment, the saturated pixel luminance is obtained from the value indicated by the saturated pixel number ratio or the threshold pixel number ratio in the luminance distribution. Instead, the saturated region radius r _s , the threshold excess region The radius r _t or the diameter may be used as an index.

For example, when the radii r _s and r _t are used, the saturated pixel luminance J ₀ in the second embodiment is calculated using the following formula (11).

Then, when the saturated pixel luminance J ₀ in the third embodiment is calculated using the radii r _s and r _t , it is calculated using the following formula (12).

  According to the third embodiment, even when the value of σ is unknown, the optimum exposure time TeB can be determined, and the adjustment time of the exposure time can be shortened by about 2 to 3 times.

  Next, processing of the microinjection apparatus 300 shown in the third embodiment will be described. FIG. 16 is a flowchart showing processing of the microinjection apparatus shown in the third embodiment.

First, data such as the combination of the discharge pressure Pi and the discharge pressure duration Ti, the discharge pressure maximum value Pi _MAX , the discharge pressure duration maximum value Ti _{MAX and the} like are input to the microinjection apparatus 300 (step) S300).

  The various data input in step S300 is stored in the storage unit 101a (step S301). The ejection control unit 101b sets the initial exposure time Te1 of the high sensitivity CCD camera 106 (step S302).

Then, the discharge control unit 101b acquires the discharge pressure Pi _MAX and the discharge time Ti _MAX from the storage unit 101a, and outputs a discharge command based on the acquired Pi _MAX and Ti _MAX (step S303).

Thereafter, the image acquisition unit 101c acquires a fluorescent image (step S304), and the exposure time control unit 101d acquires the maximum pixel luminance J _MAX from the acquired image (step S305).

Then, the exposure time control unit 101d compares the J _MAX acquired in step S305 with the J _HIGH stored in the storage unit 101a, and when J _MAX exceeds J _HIGH (step S306, Yes), the threshold value. The pixel number ratio calculation unit 101f calculates the threshold pixel number ratio n _t and outputs it to the saturated pixel luminance calculation unit 101e. Further, the saturated pixel luminance calculation unit 101e calculates the saturated pixel number ratio n _s (step S307).

Thereafter, the saturated pixel luminance calculation unit 101e uses the equation (10), and calculates the saturated pixel brightness _{J 0} (step S308). Then, multiplying the C1 / _{J 0} to the initial exposure time Te1 (step S309).

Then, the ejection control unit 101b determines whether or not the exposure time Te2 calculated in step S309 is less than the lower limit Te _{LOW of the} exposure time, and when Te2 is not less than Te _LOW (No in step S310), step S303. Migrate to

On the other hand, if Te2 is less than Te _LOW (step S310, Yes), it is determined that the discharge volume exceeds the allowable calibration range, and an error is interrupted (step S311).

If J _MAX does not exceed J _HIGH from the comparison result shown in step S306 (No in step S306), the process proceeds to step S312 and is stored in the J _MAX acquired in step S305 and the storage unit 101a. Compare J _LOW .

From the comparison result, when J _MAX exceeds J _LOW (step S312, No), the discharge calibration determines the exposure time Te1 set in step S302 as the optimum exposure time TeB (step S316).

On the other hand, when J _MAX does not exceed J _LOW (step S312, Yes), the exposure time control unit 101d determines the ratio of C1 = (J _H1 + J _L1 ) / 2 and J _MAX to the exposure time set in step S302. Multiply by (C1 / J _MAX ) (step S313).

Then, the discharge control unit 101b determines whether or not the exposure time calculated in step S313 (for example, Te2 is less than the upper limit Te _{HIGH of the} exposure time, and if Te2 does not exceed Te _HIGH (step S314, No), the process proceeds to step S303.

On the other hand, when Te2 is less than Te _HIGH (step S314, Yes), an error is interrupted on the assumption that the discharge volume is below the allowable calibration range (step S315).

According to this flowchart, the saturation pixel luminance J ₀ can be calculated for an arbitrary value of σ, the maximum pixel luminance J _MAX is adjusted to a value within the range defined by J _HIGH and J _{LOW , and} ejection is performed. The allowable range of calibration can be expanded to such a level that there is no practical problem.

  By the way, the microinjection apparatus 100 shown in Embodiment 1 captures a plurality of fluorescent images within 0.5 seconds. From this, if a plurality of situations immediately after the introduction of the introduced substance are photographed, a plurality of fluorescent images can be acquired before the introduced substance is diffused widely.

Therefore, the microinjection apparatus 400 shown in Example 4 below acquires a plurality of fluorescent images while changing the exposure time immediately after the introduction of the introduced substance, and acquires each maximum pixel luminance J _MAX from the acquired plurality of fluorescent images. and, from among the _{J MAX acquired,} selecting a _{J MAX} with a value range defined by _{J HIGH} and _{J LOW,} the optimum exposure time TeB the exposure time corresponding to the selected _{J MAX.}

  The microinjection apparatus 400 shown in the fourth embodiment has substantially the same function as that of the microinjection apparatus 100 shown in the first embodiment, and detailed description of functional units having the same functions as the microinjection apparatus 100 is omitted. To do.

  FIG. 17 is a diagram illustrating the microinjection apparatus according to the fourth embodiment. A microinjection device 400 shown in FIG. 17 includes a control unit 101, a capillary needle operation unit 102, a capillary needle 103, a petri dish 104, a microscope 105, a high-sensitivity CCD camera 106, a fluorescent filter 107, and a reflecting mirror 108. And an excitation filter 109 and an excitation light source 110.

  The control unit 101 includes a storage unit 101a, an ejection control unit 101b, an image acquisition unit 101c, an exposure time control unit 101d, and a fluorescent image selection unit 101g.

  Therefore, the difference from the microinjection apparatus 100 is that the control unit 101 has a fluorescent image selection unit 101g. Hereinafter, the process performed by the fluorescence image selection unit 101g will be described.

  First, the ejection control unit 101b sets the initial exposure time of the high sensitivity CCD camera 106 to Te40, Te41, and Te42. The longest initial exposure time is Te42, the shortest initial exposure time is Te40, and the initial exposure time between Te40 and Te42 is Te41.

  Further, a difference of 10 times or more is provided between Te40, Te41, and Te42. For example, when Te40 is set to “0.001 seconds”, Te41 and Te42 are 0.01 seconds and 0.1 seconds, respectively. And

Then, the fluorescence image selection unit 101g acquires each fluorescence image captured by Te40, Te41, and Te42 from the image acquisition unit 101c, and sets the maximum pixel luminance of the three acquired fluorescence images as J _MAX 40, J _{It is} assumed that _MAX 41 and J _MAX 42.

Then, the fluorescence image selecting section 101g selects the maximum _{J HIGH} maximum maximum pixel brightness _{J MAX} at not exceed the adjustment target from among the _{J MAX} as described above, the selected _{J MAX} and _{J MAX} 4A, J _MAX 4A is output to the exposure time control unit 101d.

Then, when the acquired J _MAX 4A exceeds J _LOW , the exposure time control unit 101d determines the initial exposure time Te4A corresponding to J _MAX 4A as the optimum exposure time TeB.

For example, if the maximum pixel brightness corresponding to J _MAX 4A is J _MAX 40, Te4A = Te40 is set as the optimal exposure time TeB, and if the maximum pixel brightness corresponding to J _MAX 4A is J _MAX 41, Te4A = Te41. Is the optimal exposure time TeB, and if the maximum pixel brightness corresponding to J _MAX 4A is J _MAX 42, Te4A = Te42 is set as the optimal exposure time TeB.

Subsequently, when the acquired J _MAX 4A does not exceed J _LOW (J _MAX 4A <J _Low ), the processing of the exposure time control unit 101d will be described. First, exposure time control unit 101d _multiplies C1 = the initial exposure time Te4A corresponding to _{J MAX 4A (J HIGH + J} LOW) / 2 and _{J MAX} 4A ratio _{(C1 / J} MAX 4A), the multiplication value “Te4A _A ” is output to the discharge controller 101b.

For example, in J _MAX 4A = “5”, J _LOW = “10”, and J _HIGH = “20”, C1 is “15”, and the value obtained by multiplying the initial exposure time Te4A by three from the ratio with J _MAX 4A. Becomes “Te4A _A ”.

As a result, when the image is taken with the exposure time “Te4A _A ”, J _MAX 4A is “15” and has a value in the range of J _HIGH and J _LOW .

Therefore, the discharge control unit 101b, when Te4A _A input by the exposure time control unit 101d is less than the upper limit _{Te HIGH} exposure time _(Te40 A _{<Te H1),} update the initial exposure time Te4A to Te4A _A discharge The command is issued again to the capillary needle operation unit 102.

On the other hand, when Te4A _A input by the exposure time control unit 101d exceeds the upper limit Te _{HIGH of the} exposure time (Te40 _A > Te _HIGH ), the discharge control unit 101b discharges the discharge control unit. 101b does not update the reference exposure time Te4A to Te4A _A , and suspends the discharge calibration because the discharge volume is below the allowable calibration range.

  Next, processing of the microinjection apparatus 400 shown in the fourth embodiment will be described. FIG. 18 is a flowchart for explaining processing of the microinjection apparatus shown in the fourth embodiment.

First, data such as the combination of the discharge pressure Pi and the discharge pressure duration Ti, the maximum discharge pressure value Pi _MAX , and the maximum discharge pressure duration Ti _MAX are input to the microinjection device 400 (step). S400).

  The various data input in step S400 is stored in the storage unit 101a (step S401). The discharge controller 101b prepares initial exposure times Te40, Te41, and Te42 that are sequentially set in the high-sensitivity CCD camera 106 (step S402).

Then, the discharge control unit 101b acquires the discharge pressure Pi _MAX and the discharge time Ti _MAX from the storage unit 101a, and outputs a discharge command based on the acquired Pi _MAX and Ti _MAX (step S403).

Thereafter, the image acquisition unit 101c sequentially sets the high-sensitivity CCD camera 106 until the initial exposure time Te40 to Te42, and repeats the acquisition of fluorescent images (step S404), and selects the acquired three images as fluorescent images. Output to the unit 101g. Then, the fluorescence image selection unit 101g acquires the maximum pixel brightness J _MAX from each acquired fluorescence image (step S405).

Then, the fluorescence image selecting section 101g selects the _{J MAX} of the maximum pixel brightness in which not to exceed the upper limit _{J HIGH} adjustment target from among the _{J MAX} (step S406, No) (step S407).

Here, in step S405, the fluorescence image selection unit 101g acquires three fluorescence images, and can select J _MAX having the maximum pixel brightness that does not exceed the upper limit J _{HIGH of the} adjustment target from among each J _MAX. If not (Yes in Step S406), the discharge calibration is interrupted on the assumption that the discharge volume exceeds the allowable calibration range (Step S408).

Next, the J _MAX selected by the fluorescent image selection unit 101g is output to the exposure time control unit 101d, and the exposure time control unit 101d determines that the acquired J _MAX exceeds J _LOW (No in step S409). Determines an initial exposure time Te4A corresponding to J _MAX as an optimum exposure time TeB (step S410).

On the other hand, when J _MAX selected by the fluorescent image selection unit 101g does not exceed J _LOW (step S409, Yes), C1 = (J _HIGH + J _LOW ) / 2 is set as the initial exposure time corresponding to J _MAX. The ratio of J _MAX (C1 / J _MAX ) is multiplied (step S411).

The ejection control unit 101b updates the exposure time to Te4A _A when the exposure time (for example, Te4A _A ) input from the exposure time control unit 101d is less than the upper limit Te _{HIGH of the} exposure time (No in step S412). Then, the discharge command is issued again to the capillary needle operation unit 102 (step S413).

On the other hand, the discharge control unit 101b, Te4A _A input by the exposure time control unit 101d is, unlike the case described above, if it exceeds the upper limit _{Te HIGH} exposure time (step S412, Yes), the discharge control unit 101b It is without updating the exposure time Te4A to Te4A _a, interrupts the discharge calibration as the ejection volume V is below the calibration tolerance (step S418).

After step S413, the image acquisition unit 101c acquires a fluorescent image (step S414), and the exposure time control unit 101d acquires the maximum pixel luminance J _MAX from the acquired image (step S415).

Then, the exposure time control unit 101d compares the J _MAX acquired in step S415 with the J _HIGH stored in the storage unit 101a, and if J _MAX exceeds J _HIGH (step S416, Yes), the step constant magnification exposure time Te4A _a set in S413 (for example, 0.8 times) multiplying (step S417). Thereafter, the process proceeds to step S413.

On the other hand, when J _MAX does not exceed J _HIGH (step S416, No), the process proceeds to step S409.

  According to this flowchart, since the three types of exposure time have a range of 100 times, even when the discharge volume is extremely large at the time of the first test discharge, it is not saturated and the maximum pixel of an appropriate size A fluorescent image having luminance can be acquired, and the allowable range of discharge calibration can be expanded to a level that causes no problem in practice.

5, 11, 12, 14, 15, 16, 17 Graph 20 Background image 21, 23, 24 Fluorescent image 22 Difference image 30, 31, 32, 40, 41, 90, 91, 92, 93 Luminance profile 50, 100, 200, 300, 400 Microinjection apparatus 50a, 103 Capillary needle 50b Fluorescent droplet 50c, 104 Petri dish 50d Medium 50e, 105 Microscope 50f, 107 Fluorescent filter 50g, 106 High-sensitivity CCD camera 50h Fluorescent reagent 50i Petri dish 101 Control unit 101a Memory Unit 101b Discharge control unit 101c Image acquisition unit 101d Exposure time control unit 101e Saturated pixel luminance calculation unit 101f Threshold pixel number ratio calculation unit 101g Fluorescent image selection unit 102 Capillary needle operation unit 108 Reflective mirror 109 Excitation filter 110 Excitation light source 11 constant table 500 and 600 two-dimensional normal distribution

Claims (3)

A fluorescent image acquisition unit that mixes a fluorescent reagent into an object to be injected into a cell, captures the fluorescent reagent by photographing the fluorescent reagent with a predetermined exposure time, and
A determination unit that acquires a luminance value of maximum pixel luminance from the fluorescent image and determines whether the luminance value is included in a predetermined range;
A luminance estimation unit that calculates a ratio between the number of pixels of the entire fluorescence image and the number of pixels in an area exceeding the upper limit of the imaging system of the fluorescence image acquisition unit, and estimates a true maximum luminance value using the calculated ratio When,
When the luminance value is not included in the predetermined range, an exposure time is determined such that the luminance value is included in the predetermined range, and the predetermined exposure time is adjusted according to the determined exposure time. And an exposure time adjustment unit for adjusting the exposure time such that the true maximum luminance value estimated by the luminance estimation unit is included in the predetermined range ;
Microinjection apparatus characterized by having a. The luminance estimation unit
The ratio and a threshold value that does not exceed the ratio are set, the ratio of the number of pixels in the region exceeding the threshold value to the entire fluorescent image is further calculated, and the true maximum luminance is calculated using the two calculated ratios. The microinjection apparatus according to claim 1 , wherein a value is calculated. The fluorescent image acquisition unit
A plurality of fluorescent images having different exposure times are acquired, and a fluorescent image including a maximum luminance value within the predetermined range is selected from the acquired plurality of fluorescent images, and the exposure time adjustment unit is configured to select a maximum of the selected fluorescent images. The microinjection apparatus according to claim 1, wherein the exposure time is adjusted based on a luminance value.

Images