JP2010148454A - Microinjection system and method for measuring anti-backflow pressure - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure in high accuracy a proper anti-backflow pressure to prevent the backflow of an objective material into a capillary needle, and enable inhibiting the leakage of the objective material from the capillary needle. <P>SOLUTION: A microinjection system functioning to apply anti-backflow pressure on a capillary needle filled with the objective material to be injected into cells is provided, including: an ejector part functioning to eject the objective material from the tip of the capillary needle; an ejection rate-calculating section functioning to calculate the ejection rate, i.e. an ejection level per unit time of the objective material ejected by the ejector part; a pressure-reducing part functioning to reduce the pressure applied on the capillary needle by the ejector part until the ejection rate calculated by the ejection rate-calculating section comes to a predetermined threshold value; and an anti-backflow-determining part functioning to determine a pressure as the anti-backflow pressure when the ejection rate calculated by the ejection rate-calculating section comes to the predetermined threshold value. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a microinjection apparatus and a backflow prevention pressure measuring method, and more particularly to an appropriate backflow prevention capable of preventing backflow of an object into the capillary needle and suppressing leakage of the object from the capillary needle. The present invention relates to a microinjection apparatus and a backflow prevention pressure measuring method capable of measuring pressure with high accuracy.

  In recent years, research has been actively conducted to observe the alteration of genetic information of cells by injecting a target such as a drug into the cell. At that time, the target such as a gene or drug is injected into the cell. As one of the methods, the microinjection method is widely used.

  In the microinjection method, a hollow needle (capillary needle) filled with a target substance such as a gene or drug solution is pierced into a cell immersed in a buffer solution in a petri dish, and the target object in the capillary needle is discharged into the cell. Then, the target is injected into the cell. When the injection of the target is finished, the capillary needle is pulled out of the cell, and the tip is immersed in a buffer solution in the petri dish.

  By the way, if the capillary needle is immersed in the buffer solution in the petri dish, the target object may flow back into the capillary needle due to a capillary phenomenon. Therefore, in general, in the microinjection method, a predetermined pressure (hereinafter referred to as “backflow prevention pressure”) for preventing the backflow of the object is applied to the capillary needle.

  However, if an excessive backflow prevention pressure is applied to the capillary needle, as shown on the left side of FIG. 14, the target object in the capillary needle leaks out, and for example, fluorescent excitation light illumination such as a mercury lamp is applied to the cell. In the case of performing the fluorescence observation to irradiate, since the fluorescent substance is mixed with the target object, as shown on the right side of FIG.

  Therefore, in the microinjection method, an appropriate backflow prevention pressure capable of preventing the backflow of the target object into the capillary needle and suppressing the leakage of the target object from the capillary needle is measured, and this is measured. It is important to add to

  As a technique for measuring the backflow prevention pressure, the relationship between the discharge amount of the target object with respect to the pulsed (intermittent) pressure (pulse discharge pressure) is obtained as an approximate line, and the backflow prevention pressure is extrapolated from this approximate line. There is something to measure. That is, as shown in FIG. 11, a plurality of pulse discharge pressures having different values are applied to the capillary needle, the target object is discharged from the tip of the capillary needle, and an image of the target object is captured by fluorescence observation. Further, the discharge amount of the target object for each pulse discharge pressure is obtained from the image of the target object by fluorescence observation. Furthermore, an approximate straight line of the discharge amount of the object for each pulse discharge pressure is generated, and a pressure at which the discharge amount becomes zero on the approximate straight line is determined as a backflow prevention pressure.

JP 2004-081084 A JP 2007-289081 A

  However, the conventional technique for measuring the backflow prevention pressure by extrapolation from the approximate straight line described above has a problem that the measurement accuracy of the backflow prevention pressure is poor. That is, as shown in FIG. 12, when there is a measurement variation in the value of the discharge amount of the object with respect to each pulse discharge pressure, the approximate straight line of the discharge amount of the object with respect to each pulse discharge pressure and the value of the true discharge amount Since they are far from each other, the backflow prevention pressure obtained by extrapolation from the approximate straight line may also deviate from the true value.

  Further, as shown in FIG. 13, the relationship between the pulse discharge pressure and the discharge amount of the object is not actually a complete linear relationship, and the relationship between the two in the region of the minute discharge amount is a non-linear relationship. Therefore, the backflow prevention pressure obtained by extrapolation from the approximate straight line may be deviated from the true value.

  The disclosed technology has been made to solve the above-described problems caused by the conventional technology, and prevents the backflow of the object into the capillary needle and suppresses the leakage of the object from the capillary needle. It is an object of the present invention to provide a microinjection apparatus and a backflow prevention pressure measuring method capable of measuring an appropriate backflow prevention pressure with high accuracy.

  In order to solve the above-described problems and achieve the object, a microinjection device disclosed in the present application, in one aspect, applies a backflow prevention pressure to a capillary needle filled with an object to be injected into a cell. A microinjection device, wherein a pressure is applied to the capillary needle to discharge the target from the tip of the capillary needle, and a discharge per unit time of the target discharged by the discharge unit A discharge rate calculation unit that calculates a discharge rate that is a quantity, and a pressure that is applied to the capillary needle by the discharge unit until the discharge rate calculated by the discharge rate calculation unit is equal to or less than a predetermined threshold value. The pressure reducing part to be reduced and the discharge rate calculated by the discharge rate calculating part are below the predetermined threshold value. The pressure at the time was, and a reverse flow preventing pressure determination unit for determining as the reverse flow preventing pressure.

  According to this aspect, it is possible to accurately measure an appropriate backflow prevention pressure capable of preventing the backflow of the target object into the capillary needle and suppressing the leakage of the target object from the capillary needle.

  In addition, in order to solve the above-described problem, a component, expression, or any combination of components of the microinjection device disclosed in the present application is applied to a method, a device, a system, a computer program, a recording medium, a data structure, and the like. It is effective for.

  According to one aspect of the microinjection apparatus disclosed in the present application, an appropriate backflow prevention pressure that prevents backflow of the target object into the capillary needle and suppresses leakage of the target object from the capillary needle is provided. There is an effect that measurement can be performed with high accuracy.

  Exemplary embodiments of a microinjection apparatus and a backflow prevention pressure measuring method disclosed in the present application will be described below in detail with reference to the accompanying drawings.

  First, the outline of the backflow prevention pressure measuring method according to the present embodiment will be described. The backflow prevention pressure measuring method according to the present embodiment is a pressure for preventing backflow of the target object into the capillary needle with respect to the capillary needle filled with the target object such as a chemical solution to be injected into the cell. It is applied to a microinjection device that applies backflow prevention pressure.

  FIG. 1 is a diagram for explaining the outline of the backflow prevention pressure measuring method according to the present embodiment. As shown in FIG. 1, in the backflow prevention pressure measuring method according to the present embodiment, first, pressure is applied to the capillary needle, and a target object such as a chemical solution containing a fluorescent substance is discharged from the tip of the capillary needle. Subsequently, the ejected target object is imaged at a predetermined time interval with a camera while being magnified by the objective lens, and the target object ejected from the tip of the capillary needle is ejected per unit time using the image of the object. A discharge rate that is a quantity is calculated.

  Subsequently, the pressure applied to the capillary needle is decreased until the calculated discharge rate is equal to or lower than a predetermined threshold value. In this case, each time the pressure applied to the capillary needle is decreased, the discharge rate of the target object discharged at the reduced pressure is calculated using the target object image in the same manner as described above. Then, the pressure applied to the capillary needle when the calculated discharge rate becomes equal to or less than a predetermined threshold is determined as the backflow prevention pressure.

  As described above, in the backflow prevention pressure measuring method according to the present embodiment, the backflow prevention pressure is not measured by extrapolation from the approximate straight line as in the past, but each time the pressure applied to the capillary needle is repeatedly reduced. In addition, the backflow prevention pressure is directly measured by calculating the discharge rate of the object discharged from the capillary needle and determining the discharge rate as a threshold value. For this reason, it is possible to accurately measure an appropriate backflow prevention pressure that can prevent backflow of the target object into the capillary needle and can suppress leakage of the target object from the capillary needle.

  Next, the configuration of the microinjection apparatus to which the backflow prevention pressure measuring method according to the present embodiment is applied will be described. FIG. 2 is a diagram illustrating a schematic configuration of the microinjection apparatus according to the present embodiment. As shown in FIG. 2, the microinjection apparatus 100 includes a capillary needle 110, a petri dish 120, a stage 130, a camera module 140, a discharge device 150, a capillary needle driving device 160, a stage driving device 170, an objective, and the like. A lens driving device 180 and a system control device 190 are included.

  The capillary needle 110 is a hollow glass needle whose tip is miniaturized, and is filled with a target such as a chemical solution to be injected into cells on the petri dish 120. The capillary needle 110 is connected to the discharge device 150, and continuously discharges the target object filled therein by the pressure applied from the discharge device 150. The capillary needle 110 is attached to a capillary needle driving device 160 and is configured to move on a horizontal plane and to advance and retreat in the longitudinal direction.

  The petri dish 120 is a flat dish-shaped transparent container, and the inside is filled with a buffer that can survive cells. The petri dish 120 is placed on the stage 130 and moves on the horizontal plane together with the stage 130.

  The stage 130 is an XY stage that is attached to the stage driving device 170 and is movable in the horizontal direction under the control of the stage driving device 170. Further, the stage 130 is configured so that the petri dish 120 can be mounted on the upper surface, and the stage 130 is manufactured from a transparent material so that the camera module 140 can take an image of the inside of the petri dish 120, or partly There is a hole for shooting.

  The camera module 140 includes an objective lens 141, an excitation light source 142, an excitation filter 143, a reflecting mirror 144, a fluorescence filter 145, and a camera 146. The objective lens 141 is a lens for obtaining an enlarged image of a part on the petri dish 120, for example, the vicinity of the tip of the capillary needle 110. The excitation light source 142 is a light source for irradiating this object with fluorescence excitation light when an object such as a chemical solution containing a fluorescent material is ejected from the tip of the capillary needle 110. The excitation filter 143 is a filter that narrows the fluorescence excitation light emitted from the excitation light source 142 to a predetermined wavelength and sends it to the reflecting mirror 144.

  The reflecting mirror 144 is a mirror for reflecting the image obtained by the objective lens 141 toward the fluorescent filter 145 and reflecting the fluorescence excitation light emitted from the excitation light source 142 toward the petri dish 120. The fluorescence filter 145 is a filter that removes light having a wavelength other than the fluorescence wavelength from the image obtained by the objective lens 141 and forms this image on the video element of the camera 146.

  The camera 146 is a device that captures an image reflected by the reflecting mirror 144, and outputs the captured image to the system control device 190. The camera 146, for example, captures an enlarged image near the tip of the capillary needle 110 obtained by the objective lens 141, that is, an image of a target object ejected from the tip of the capillary needle 110, and outputs it to the system control device 190.

  The discharge device 150 is a device that applies pressure to the capillary needle 110 in accordance with an instruction from the system control device 190 and discharges a target object from the tip of the capillary needle 110. The capillary needle drive device 160 is a device that drives the capillary needle 110 in accordance with an instruction from the system control device 190. For example, the capillary needle driving device 160 horizontally moves the capillary needle 110 so that the tip of the capillary needle 110 is positioned vertically above the objective lens 141 when measuring the backflow prevention pressure.

  The stage driving device 170 is a device that controls the stage 130 in accordance with instructions from the system control device 190. For example, the stage driving device 170 horizontally moves the stage 130 so that the target discharged from the tip of the capillary needle 110 is positioned vertically above the objective lens 141 when measuring the backflow prevention pressure.

  The objective lens driving device 180 is a device that drives the objective lens 141 in accordance with an instruction from the system control device 190. For example, the objective lens driving device 180 horizontally moves the objective lens 141 so that the objective lens 141 is positioned vertically below the capillary needle 110.

  The system control device 190 is a control device that controls the microinjection device 100 as a whole, and controls the operations of the camera 146, the ejection device 150, the capillary needle drive device 160, the stage drive device 170, and the objective lens drive device 180.

  Under such a configuration, in the microinjection device 100 according to the first embodiment, when measuring the backflow prevention pressure, the capillary needle driving device 160 moves the capillary needle 110 in accordance with an instruction from the system control device 190 to drive the stage. The device 170 moves the stage 130, the objective lens driving device 180 moves the objective lens, and the ejection device 150 applies pressure to the capillary needle 110 to buffer the target object from the tip of the capillary needle 110 in the petri dish 120. Discharge into liquid.

  At this time, in the microinjection apparatus 100, the camera 146 images the target object discharged from the capillary needle 110 at a predetermined time interval, and the system controller 190 uses the image of the target object to detect the target object discharged from the capillary needle 110. The discharge rate of the target object discharged from the tip is calculated.

  Subsequently, in the microinjection device 100, the system control device 190 instructs the discharge device 150 to decrease the pressure applied to the capillary needle 110 until the discharge rate becomes equal to or lower than a predetermined threshold value. In this case, each time the pressure applied by the ejection device 150 to the capillary needle 110 is decreased, the system control device 190 calculates the ejection rate of the target object ejected from the tip of the capillary needle 110 with the decreased pressure. . Then, the system control device 190 determines the pressure applied to the capillary needle 110 by the discharge device 150 when the discharge rate is equal to or lower than a predetermined threshold as the backflow prevention pressure.

  Thus, in the microinjection apparatus 100 according to the present embodiment, the backflow prevention pressure is not measured by extrapolation from the approximate straight line as in the conventional example, but each time the pressure applied to the capillary needle 110 is decreased. In addition, the backflow prevention pressure is directly measured by calculating the discharge rate of the object discharged from the capillary needle 110 and determining the discharge rate as a threshold value. For this reason, it is possible to measure an appropriate backflow prevention pressure that can prevent the backflow of the target object into the capillary needle and suppress the leakage of the target object from the capillary needle with higher accuracy than in the past. .

  Next, the configuration of the system control device 190 shown in FIG. 2 will be specifically described. FIG. 3 is a block diagram showing a configuration of the system control apparatus 190 shown in FIG. As shown in FIG. 3, the system control device 190 is connected to the ejection device 150, the capillary needle driving device 160, the stage driving device 170, the objective lens driving device 180, the camera 146, and the bus. .

  Further, the system control device 190 includes a discharge device control unit 191, a drive device control unit 192, an imaging unit 193, an image storage unit 194, a discharge rate calculation condition storage unit 195, a discharge rate calculation unit 196, a discharge A rate determination unit 197, a decompression unit 198, and a backflow prevention pressure determination unit 199 are included.

  The discharge device control unit 191 is a processing unit that controls the discharge device 150 to apply pressure to the capillary needle 110 and discharge a target object from the tip of the capillary needle 110. Further, the discharge device control unit 191 applies the reduced pressure reduced by the decompression unit 198 to the capillary needle 110, and discharges the target object from the tip of the capillary needle 110 with the reduced pressure. Instruct the device 150. Note that the pressure applied to the capillary needle 110 is not a pulsed (intermittent) pressure but a continuous pressure.

  The drive device control unit 192 is a processing unit that moves the capillary needle 110, the stage 130, and the objective lens 141 by controlling the capillary needle drive device 160, the stage drive device 170, and the objective lens drive device 180, respectively.

  Specifically, the drive device controller 192 controls the capillary needle drive device 160 at the start of measurement of the backflow prevention pressure so that the tip of the capillary needle 110 is positioned vertically above the objective lens 141. 110 is moved horizontally. Further, the driving device control unit 192 controls the stage driving device 170 at the start of the measurement of the backflow prevention pressure so that the target discharged from the tip of the capillary needle 110 is positioned vertically above the objective lens 141. The stage 130 is moved horizontally. Further, the driving device control unit 192 controls the objective lens driving device 180 at the start of measurement of the backflow prevention pressure, and horizontally moves the objective lens 141 so that the objective lens 141 is positioned vertically below the capillary needle 110. .

  Further, when the drive device control unit 192 receives a notification from the decompression unit 198 that the pressure applied to the capillary needle 110 has decreased, the drive device control unit 192 sets the capillary needle 110 and the objective lens 141 or the stage 130 to the current position. Move horizontally to a different position. In other words, the driving device control unit 192 detects the position of the target already discharged from the tip of the capillary needle 110 at the pressure before the pressure reduction by the pressure reduction unit 198 and the pressure after the pressure reduction by the pressure reduction unit 198 The capillary needle 110 and the like are horizontally moved so that the position of the target object to be discharged is shifted. As a result, an object discharged from the tip of the capillary needle 110 at a pressure before reduction by the decompression unit 198 and an object discharged from the tip of the capillary needle 110 at a pressure after reduction by the pressure reduction unit 198 are contained in the petri dish 120. It is possible to prevent the image quality of the image obtained by the objective lens 141 from deteriorating by overlapping at the same position in the buffer solution. As a result, it is possible to prevent the image quality of the target image captured by the camera 146 from being degraded.

  The imaging unit 193 is a processing unit that causes the camera 146 to image cells on the petri dish 120 and stores the captured image in the image storage unit 194 when microinjection is performed. In addition, the imaging unit 193 causes the camera 146 to capture an image of the target ejected from the tip of the capillary needle 110 at a predetermined time interval when measuring the backflow prevention pressure, and stores the captured image in the image storage unit 194. It is a processing unit. Specifically, the imaging unit 193 performs an image of the target immediately after the target is discharged from the tip of the capillary needle 110 and the capillary each time a discharge rate calculation process is executed by a discharge rate calculation unit 196 described below. The camera 146 is caused to capture the image of the target object after the predetermined time interval has passed immediately after the target object is discharged from the tip of the needle 110. Hereinafter, a predetermined time interval at which the camera 146 captures an image of the object is referred to as “imaging interval time”.

  The image storage unit 194 is a storage unit that stores an image captured by the camera 146. The image stored in the image storage unit 194 is used for the discharge rate calculation by the discharge rate calculation unit 196 described later.

  The discharge rate calculation condition storage unit 195 is a storage unit that stores discharge rate calculation conditions. Specifically, the discharge rate calculation condition storage unit 195 stores the exposure time of the camera 146, the concentration of the fluorescent substance in the target object filled in the capillary needle 110, the total luminance of the fluorescent substance measured in advance, and its discharge amount. Is stored as a discharge rate calculation condition.

  The discharge rate calculation unit 196 uses the image stored in the image storage unit 194 and the discharge rate calculation condition stored in the discharge rate calculation condition storage unit 195 to unit time the target discharged from the tip of the capillary needle 110. A processing unit that calculates a discharge rate that is a discharge amount per unit. The discharge rate calculation unit 196 is discharged from the tip of the capillary needle 110 at the pressure reduced by the decompression unit 198 every time a notification that the pressure applied to the capillary needle 110 has been reduced is received from the decompression unit 198. The discharge rate of the target object is calculated repeatedly. Here, the discharge rate calculation processing by the discharge rate calculation unit 196 will be specifically described with reference to FIG. FIG. 6 is a diagram for explaining the discharge rate calculation processing.

  As shown in FIG. 6, first, the discharge rate calculation unit 196 first captures an image of the target image captured by the camera 146 immediately after the target object is discharged from the tip of the capillary needle 110 (hereinafter, “the target object immediately after discharge” Image A) and an image of the object imaged by the camera 146 after the imaging interval time has elapsed immediately after the target object is ejected (hereinafter referred to as an “image of the object object after the imaging interval time has elapsed”). .) B is read from the image storage unit 194. Each time the discharge rate calculation unit 196 executes the discharge rate calculation process, the discharge rate calculation unit 196 instructs the imaging unit 193 to display the target image A immediately after the discharge and the target image B after the imaging interval time has elapsed. The image is newly captured and these captured images are stored in the image storage unit 194.

  Subsequently, the discharge rate calculation unit 196 calculates the luminance sum of the difference image C between the image A of the object immediately after the discharge and the image B of the object after the imaging interval time has elapsed.

Hereinafter, a method for calculating the luminance sum of the difference image C between the image A of the object immediately after ejection and the image B of the object after the imaging interval time has elapsed will be described. That is, the horizontal pixel position in the image captured by the camera 146 is x, the vertical pixel position in the image is y, and the luminance at the pixel position (x, y) of the target image A immediately after ejection is FL1. If the luminance at the pixel position (x, y) of the target image B after the lapse of the imaging interval time is FL2 (x, y), the total luminance I of the difference image C is expressed by the following equation. expressed.
I = Σ (FL2 (x, y) −FL1 (x, y)) (1)

Subsequently, the discharge rate calculation unit 196 calculates the discharge amount of the target object based on the luminance sum I of the difference image C calculated as described above. Specifically, the discharge rate calculation unit 196 calculates the discharge amount of the target object using the total luminance of the difference image C and the discharge rate calculation condition stored in the discharge rate calculation condition storage unit 195. That is, the exposure time of the camera 146 included in the discharge rate calculation condition is Te, the concentration of the fluorescent material of the target object filled in the capillary needle 110 is ρ, the luminance total of the fluorescent material measured in advance and the discharge amount thereof When the conversion coefficient is α, the discharge amount V of the target object is expressed by the following equation.
V = α · I / (ρ · Te) (2)

  Finally, the discharge rate calculation unit 196 calculates the discharge rate of the target discharged from the tip of the capillary needle 110 by dividing the discharge amount of the target calculated as described above by the imaging interval time. . The discharge rate calculation unit 196 notifies the discharge rate determination unit 197 of the discharge rate calculated in this way.

  Returning to the explanation of FIG. 3, when the discharge rate determination unit 197 receives a notification of the discharge rate of the target object discharged from the tip of the capillary needle 110 from the discharge rate calculation unit 196, the discharge rate is determined to be a predetermined value. It is a process part which determines whether it is below a threshold value. When the discharge rate of the target discharged from the tip of the capillary needle 110 is larger than a predetermined threshold, the discharge rate determination unit 197 notifies the decompression unit 198 to that effect while discharging from the tip of the capillary needle 110 When the discharge rate of the target object is equal to or lower than a predetermined threshold value, the fact is notified to the backflow prevention pressure determining unit 199.

  Here, the predetermined threshold is preferably 0.01 to 1 pl / sec. This is because if the discharge rate is less than 0.01 pl, there is a risk that the target will flow backward into the capillary needle 110, while if the discharge rate is greater than 1 pl / sec, the capillary needle This is because an excessive leakage of the object may occur from the tip of 110.

  The decompression unit 198 is a processing unit that reduces the pressure applied to the capillary needle 110 until the ejection rate calculated by the ejection rate calculation unit 196 is equal to or lower than a predetermined threshold value. That is, when the discharge rate determination unit 197 determines that the discharge rate of the target discharged from the tip of the capillary needle 110 is greater than a predetermined threshold, the decompression unit 198 is applied to the capillary needle 110. Reduce pressure. Every time the pressure applied to the capillary needle 110 is reduced, the decompression unit 198 stores the reduced pressure in a predetermined storage unit (not shown).

  Further, when the pressure applied to the capillary needle 110 is reduced, the decompression unit 198 notifies the discharge device control unit 191 of the reduced pressure and discharges the reduced pressure to the capillary needle 110. Instructs the apparatus control unit 191. The decompression unit 198 also notifies the discharge rate calculation unit 196 that the pressure applied to the capillary needle 110 has been reduced, and discharges the target object discharged from the tip of the capillary needle 110 with the reduced pressure. Let the rate be calculated. Further, the decompression unit 198 notifies the drive device control unit 192 that the pressure applied to the capillary needle 110 has been reduced, and controls the drive device control unit 192 to control the capillary needle 110, the stage 130, and the objective lens 141. Is moved to a predetermined position.

  The backflow prevention pressure determining unit 199 determines a pressure applied to the capillary needle 110 as a backflow prevention pressure when the discharge rate calculated by the discharge rate calculation unit 196 is equal to or less than a predetermined threshold. It is. That is, when the notification that the discharge rate of the target discharged from the tip of the capillary needle 110 is equal to or less than a predetermined threshold is received from the discharge rate determination unit 197, the backflow prevention pressure determination unit 199 includes the pressure reduction unit 198. The reduced pressure stored in the predetermined storage unit is determined as the backflow prevention pressure.

  Next, a processing procedure of backflow prevention pressure measurement processing by the microinjection apparatus 100 according to the first embodiment will be described. FIG. 4 is a flowchart illustrating a processing procedure of backflow prevention pressure measurement processing by the microinjection apparatus 100 according to the first embodiment. At the start of the backflow prevention pressure measurement process, the drive device control unit 192 controls the capillary needle drive device 160 and the stage drive device 170 so that the tip of the capillary needle 110 is placed in the buffer solution filled in the petri dish 120. It is assumed that the capillary needle 110 and the stage 130 are horizontally moved so as to be immersed. In addition, the driving device control unit 192 controls the objective lens driving device 180 at the start of measurement of the backflow prevention pressure, and horizontally moves the objective lens 141 so that the objective lens 141 is positioned vertically below the capillary needle 110. It shall be.

  As shown in FIG. 4, the ejection device controller 191 of the microinjection device 100 controls the ejection device 150 to apply an initial pressure to the capillary needle 110 and eject the target from the tip of the capillary needle 110 ( Step S101).

  Subsequently, the driving device control unit 192 controls the stage driving device 170 to horizontally move the stage 130 to a position different from the current position (step S102). In this process, the driving device controller 192 may control the capillary needle driving device 160 and the objective lens driving device 180 to horizontally move the capillary needle 110 and the objective lens 141 to a position different from the current position. .

  Subsequently, the discharge rate calculation unit 196 starts a discharge rate calculation process (step S103). That is, the discharge rate calculation unit 196 uses the image stored in the image storage unit 194 and the discharge rate calculation condition stored in the discharge rate calculation condition storage unit 195 to detect the target object discharged from the tip of the capillary needle 110. The discharge rate is calculated, and the calculated discharge rate is notified to the discharge rate determination unit 197. The discharge rate calculation process executed by the discharge rate calculation unit 196 will be described in detail later with reference to FIG.

  Subsequently, the discharge rate determination unit 197 determines whether or not the discharge rate is equal to or lower than a predetermined threshold (step S104). When the discharge rate determination unit 197 determines that the discharge rate is greater than the predetermined threshold (step S104: No), the decompression unit 198 decreases the pressure applied to the capillary needle 110 by a certain amount, The reduced pressure is stored in a predetermined storage unit (step S105), and the process returns to step S102. In the process of step S <b> 105, the decompression unit 198 notifies the discharge device control unit 191 of the reduced pressure, and instructs the discharge device control unit 191 to apply the reduced pressure to the capillary needle 110. The discharge device control unit 191 controls the discharge device 150 in accordance with an instruction from the decompression unit 198, applies the pressure reduced by the decompression unit 198 to the capillary needle 110, and the capillary needle 110 with this reduced pressure. The target object is ejected from the tip.

  In this manner, in this embodiment, the pressure applied to the capillary needle 110 is reduced and the discharge rate is calculated until the discharge rate of the target object discharged from the tip of the capillary needle 110 becomes a predetermined threshold value or less. Repeated (see step S102 to step S105).

  On the other hand, when the discharge rate determination unit 197 determines that the discharge rate is equal to or lower than the predetermined threshold (step S104: Yes), the backflow prevention pressure determination unit 199 performs the predetermined storage unit by the pressure reduction unit 198 in step S105. Is determined as the backflow prevention pressure (step S106), and the backflow prevention pressure measurement process is terminated.

  Next, with reference to FIG. 5, the processing procedure of the discharge rate calculation process (step S103) by the microinjection apparatus 100 according to the first embodiment will be described. FIG. 5 is a flowchart illustrating a processing procedure of a discharge rate calculation process performed by the microinjection apparatus 100 according to the first embodiment.

  As illustrated in FIG. 5, the imaging unit 193 of the microinjection apparatus 100 causes the camera 146 to capture an image of the object immediately after ejection (step S201). Subsequently, the imaging unit 193 stores the captured image in the image storage unit 194 (step S202).

  Subsequently, the imaging unit 193 determines whether or not the imaging interval time has passed immediately after the target object is ejected from the tip of the capillary needle 110 (step S203). If it is determined that the imaging interval time has passed immediately after the target object is ejected from the tip of the capillary needle 110 (step S203: Yes), the imaging unit 193 causes the camera 146 to capture an image of the target object after the imaging time has elapsed. (Step S204). Subsequently, the imaging unit 193 stores the captured image in the image storage unit 194 (step S205).

  Subsequently, the discharge rate calculation unit 196 reads the image of the target object immediately after the discharge and the image of the target object after the imaging interval time has elapsed from the image storage unit 194 (step S206). Subsequently, the ejection rate calculation unit calculates the total luminance of the difference image between the target image immediately after the ejection and the target image after the imaging interval time has elapsed (step S207).

  Subsequently, the discharge rate calculation unit 196 calculates the discharge amount of the target object based on the calculated luminance sum of the difference images (step S208). Specifically, the discharge rate calculation unit 196 calculates the discharge amount of the target object using the total luminance of the difference image C and the discharge rate calculation condition stored in the discharge rate calculation condition storage unit 195.

  Subsequently, the discharge rate calculation unit 196 calculates the discharge rate of the target discharged from the tip of the capillary needle 110 by dividing the calculated discharge amount of the target by the imaging interval time (step S209). The discharge rate calculation process ends.

  As described above, the microinjection apparatus 100 according to the first embodiment calculates the discharge rate of the target object discharged from the capillary needle 110 each time the pressure applied to the capillary needle 110 is reduced, and the calculation. The backflow prevention pressure is directly measured by determining the threshold value of the discharged rate. Therefore, compared with the conventional method of measuring the backflow prevention pressure by extrapolation from the approximate straight line, the backflow of the target object into the capillary needle is prevented and the leakage of the target object from the capillary needle is suppressed. Therefore, it is possible to measure an appropriate backflow prevention pressure with high accuracy.

  Further, in the microinjection apparatus 100 according to the first embodiment, the decompression unit 198 reduces the pressure applied to the capillary needle 110 by a certain amount, so that it is necessary to separately provide a processing unit for calculating the pressure decrease amount. The device configuration can be simplified.

  In the first embodiment, the example in which the pressure reducing unit 198 reduces the pressure applied to the capillary needle 110 by a certain amount has been described. However, the pressure reducing unit 198 is configured to change the amount of decrease in the pressure applied to the capillary needle 110. May be. Accordingly, in the second embodiment, a microinjection device configured to change the amount of decrease in pressure applied to the capillary needle 110 will be described.

  First, the configuration of the system control device included in the microinjection apparatus according to the second embodiment will be described. FIG. 7 is a block diagram illustrating a configuration of a system control apparatus included in the microinjection apparatus according to the second embodiment. In the following description, parts having the same functions as the constituent parts shown in FIG. The schematic configuration of the microinjection apparatus according to the second embodiment is the same as the schematic configuration shown in FIG.

  As illustrated in FIG. 7, the system control device 290 includes a decompression unit 298, a post-decompression pressure storage unit 291, and an approximate straight line generation unit 292, instead of the decompression unit 198 included in the system control device 190 illustrated in FIG. 3. , A zero pressure calculation unit 293 and an average value calculation unit 294 are newly provided.

  The decompression unit 298 reduces the pressure applied to the capillary needle 110 until the ejection rate calculated by the ejection rate calculation unit 196 is equal to or lower than a predetermined threshold, similarly to the decompression unit 198 shown in FIG. It is a processing unit. In addition, the decompression unit 298 according to the second embodiment acquires the number of times the discharge rate calculation unit 196 calculates the discharge rate until the discharge rate calculated by the discharge rate calculation unit 196 is equal to or less than a predetermined threshold. The amount of decrease in the pressure applied to the capillary needle 110 is changed according to the obtained number of times of calculation of the discharge rate.

  In other words, when the number of times the discharge rate calculation unit 196 calculates the discharge rate is 1, the decompression unit 298 decreases the pressure applied to the capillary needle 110 by a certain amount. On the other hand, when the number of discharge rate calculations by the discharge rate calculation unit 196 is two or more times, the decompression unit 298 adds a pressure average value (described later) calculated by the average value calculation unit 294 to the capillary needle 110 next time. At the same time, the pressure applied to the capillary needle 110 is reduced to the pressure average value. Such a decompression process by the decompression unit 298 will be described in detail later.

  Further, each time the pressure applied to the capillary needle 110 is reduced, the decompression unit 298 reduces the reduced pressure, and the discharge rate of the target object discharged from the tip of the capillary needle 110 with the reduced pressure. Are stored in the post-depressurization pressure storage unit 291 in association with each other.

  The post-depressurization pressure storage unit 291 is a storage unit that stores the pressure after the decrease by the depressurization unit 298 and the discharge rate of the target object discharged from the tip of the capillary needle 110 with the decreased pressure. That is, the post-depressurization pressure storage unit 291 stores each pressure applied to the capillary needle 110 so far and the discharge rate for each pressure in association with each other.

  The approximate straight line generation unit 292 acquires each pressure applied to the capillary needle 110 up to this time and the discharge rate for each pressure from the pressure storage unit 291 after depressurization, and is applied to the capillary needle 110 up to this time. An approximate straight line of the discharge rate for each pressure is generated. The zero pressure calculation unit 293 calculates a zero pressure that is a pressure at which the discharge rate becomes zero on the approximate line generated by the approximate line generation unit 292.

  The average value calculation unit 294 acquires the pressure applied to the capillary needle 110 this time from the post-depressurization pressure storage unit 291, and the average value of the acquired pressure and the zero pressure calculated by the zero pressure calculation unit 293 (Hereinafter referred to as “pressure average value”). The average value calculation unit 294 notifies the pressure reduction unit 298 of the calculated pressure average value.

  Here, with reference to FIG. 10, the decompression process by the decompression unit 298 will be specifically described. FIG. 10 is a diagram for explaining the decompression process. In the example illustrated in FIG. 10, it is assumed that the discharge rate is equal to or lower than a predetermined threshold when the discharge rate calculation unit 196 calculates the discharge rate for the fourth time.

  As shown in FIG. 10, when the discharge rate calculation unit 196 calculates the discharge rate once, the decompression unit 298 reduces the pressure applied to the capillary needle 110 by a certain amount. Note that the pressure after being reduced by a certain amount by the decompression unit 298, that is, the pressure applied to the capillary needle 110 when the number of times the ejection rate is calculated by the ejection rate calculation unit 196 is the second time is P2.

  In addition, when the number of times the discharge rate is calculated by the discharge rate calculation unit 196 is 2, the decompression unit 298 reduces the pressure P2 applied to the capillary needle 110 this time (second time) to the pressure average value AV1. .

  Specifically, when the number of times of calculation of the discharge rate by the discharge rate calculation unit 196 is two times, first, the approximate straight line generation unit 292 first discharges the capillary needle 110 with respect to each pressure applied up to the second time. An approximate straight line L1 is generated. Next, the zero pressure calculation unit 293 calculates a zero pressure PL1, which is a pressure at which the discharge rate becomes zero, on the approximate straight line L1. Next, the average value calculation unit 294 calculates the pressure average value AV1 (= (P2 + PL1) between the pressure P2 applied to the capillary needle 110 this time (second time) and the zero pressure PL1 calculated by the zero pressure calculation unit 293. ) / 2) is calculated. Finally, the decompression unit 298 sets the pressure average value AV1 calculated by the average value calculation unit 294 to the pressure P3 to be applied to the capillary needle 110 next time (third time), and to the capillary needle 110. The pressure P2 applied this time (second time) is reduced to the pressure average value AV1 (= P3).

  Further, when the number of times of calculation of the discharge rate by the discharge rate calculation unit 196 is 3, the decompression unit 298 decreases the pressure P3 applied to the capillary needle 110 this time (third time) to the pressure average value AV2. .

  Specifically, when the number of times of calculation of the discharge rate by the discharge rate calculation unit 196 is 3, first, the approximate line generation unit 292 first discharges the capillary needle 110 with respect to each pressure applied up to the third time. An approximate straight line L2 is generated. Next, the zero pressure calculation unit 293 calculates a zero pressure PL2, which is a pressure at which the discharge rate becomes zero, on the approximate straight line L2. Next, the average value calculation unit 294 calculates the pressure average value AV2 (= (P3 + PL2) between the pressure P3 applied to the capillary needle 110 this time (third time) and the zero pressure PL2 calculated by the zero pressure calculation unit 293. ) / 2) is calculated. Finally, the decompression unit 298 sets the pressure average value AV2 calculated by the average value calculation unit 294 to the pressure P4 to be applied to the capillary needle 110 next time (fourth time), and to the capillary needle 110. The pressure P3 applied this time (third time) is reduced to the pressure average value AV2 (= P4).

  Next, a processing procedure of backflow prevention pressure measurement processing by the microinjection apparatus 200 according to the second embodiment will be described. FIG. 8 is a flowchart illustrating a processing procedure of backflow prevention pressure measurement processing by the microinjection apparatus 200 according to the second embodiment. Here, the description of the same processing procedure (step S301 to step S304, step S306) as the processing procedure shown in FIG. 4 is omitted.

  As shown in FIG. 8, when the discharge rate determination unit 197 determines that the discharge rate is greater than a predetermined threshold (step S304: No), the decompression unit 298 starts the decompression process (step S305). When the decompression process ends, the decompression unit 298 returns the process to step S302.

  Next, with reference to FIG. 9, the processing procedure of the decompression process by the microinjection apparatus 200 according to the second embodiment will be described. FIG. 9 is a flowchart illustrating a processing procedure of a decompression process performed by the microinjection apparatus 200 according to the second embodiment.

  As shown in FIG. 9, the decompression unit 298 of the microinjection apparatus 200 acquires the number of times the ejection rate is calculated by the ejection rate calculation unit 196 (step S401). Subsequently, the decompression unit 298 determines whether or not the number of times of calculation of the discharge rate by the discharge rate calculation unit 196 is two or more (step S402).

  If it is determined that the number of discharge rate calculations by the discharge rate calculation unit 196 is one (step S402: No), the decompression unit 298 decreases the pressure applied to the capillary needle 110 by a certain amount (step S403). . Thereafter, the pressure reducing unit 298 associates the reduced pressure with the discharge rate of the target object discharged from the tip of the capillary needle 110 with the reduced pressure and stores it in the post-reduced pressure storage unit 291 (step S408). ), And the decompression process ends.

  On the other hand, if it is determined that the number of discharge rate calculations by the discharge rate calculation unit 196 is two or more (step S402: Yes), the decompression unit 298 proceeds to step S404.

  In step S404, the approximate straight line generation unit 292 generates an approximate straight line of the discharge rate for each pressure applied to the capillary needle 110 so far (step S404). Specifically, the approximate straight line generation unit 292 acquires each pressure applied to the capillary needle 110 so far and the discharge rate for each pressure from the post-depressurization pressure storage unit 291, and outputs the pressure to the capillary needle 110. An approximate straight line of the discharge rate for each pressure applied up to this time is generated.

  Subsequently, the zero pressure calculation unit 293 calculates the zero pressure on the approximate line generated by the approximate line generation unit 292 (step S405). Subsequently, the average value calculation unit 294 calculates a pressure average value that is an average value of the pressure applied to the capillary needle 110 this time and the zero pressure calculated by the zero pressure calculation unit 293 (step S406). . Specifically, the average value calculation unit 294 acquires the pressure applied to the capillary needle 110 this time from the post-depressurization pressure storage unit 291, and the acquired pressure and the zero pressure calculated by the zero pressure calculation unit 293. The average value with the pressure is calculated.

  Subsequently, the decompression unit 298 sets the pressure average value calculated by the average value calculation unit 294 to a pressure to be applied to the capillary needle 110 next time, and also applies the pressure applied to the capillary needle 110 this time. Decrease to the average value (step S407). Thereafter, the pressure reducing unit 298 associates the reduced pressure with the discharge rate of the target object discharged from the tip of the capillary needle 110 with the reduced pressure and stores it in the post-reduced pressure storage unit 291 (step S408). ), And the decompression process ends.

  As described above, the microinjection apparatus 200 according to the second embodiment is similar to the first embodiment in that the objective in the capillary needle is compared to the conventional method of measuring the backflow prevention pressure by extrapolation from the approximate straight line. An appropriate backflow prevention pressure capable of preventing the backflow of the object and suppressing the leakage of the object from the capillary needle can be measured with high accuracy. Moreover, in the microinjection apparatus 200 according to the second embodiment, when the number of times of calculation of the discharge rate is two times or more, the pressure applied this time to the capillary needle 110 is reduced to the pressure average value. Rather than decreasing the pressure applied to the capillary needle 110 by a certain amount, the measurement time of the backflow prevention pressure can be shortened.

It is a figure for demonstrating the outline of the backflow prevention pressure measuring method which concerns on Example 1. FIG. It is a figure which shows schematic structure of the microinjection apparatus which concerns on Example 1. FIG. It is a block diagram which shows the structure of the system control apparatus shown in FIG. 3 is a flowchart illustrating a processing procedure of backflow prevention pressure measurement processing by the microinjection apparatus according to the first embodiment. 3 is a flowchart illustrating a processing procedure of a discharge rate calculation process performed by the microinjection apparatus according to the first embodiment. It is a figure for demonstrating a discharge rate calculation process. It is a block diagram which shows the structure of the system control apparatus which the microinjection apparatus which concerns on Example 2 has. It is a flowchart which shows the process sequence of the backflow prevention pressure measurement process by the microinjection apparatus which concerns on Example 2. FIG. 6 is a flowchart illustrating a processing procedure of a decompression process performed by a microinjection apparatus according to a second embodiment. It is a figure for demonstrating pressure reduction processing. It is a figure for demonstrating the conventional backflow prevention pressure measuring method. It is a figure for demonstrating the problem in the conventional backflow prevention pressure measuring method. It is a figure for demonstrating the problem in the conventional backflow prevention pressure measuring method. It is a figure which shows the state which the target object in the capillary needle leaked out.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Microinjection apparatus 110 Capillary needle 120 Petri dish 130 Stage 140 Camera module 141 Objective lens 142 Excitation light source 143 Excitation filter 144 Reflection mirror 145 Fluorescence filter 146 Camera 150 Discharge apparatus 160 Capillary needle drive apparatus 170 Stage drive apparatus 180 Objective lens drive apparatus 190 System Control device 191 Discharge device control unit 192 Drive device control unit 193 Imaging unit 194 Image storage unit 195 Discharge rate calculation condition storage unit 196 Discharge rate calculation unit 197 Discharge rate determination unit 198 Decompression unit 199 Backflow prevention pressure determination unit 200 Microinjection device 290 System controller 291 Pressure reduction storage unit 292 Approximate straight line generation unit 293 Zero pressure calculation unit 294 Average value calculation unit 298 Pressure reduction unit

Claims (5)

A microinjection device that applies a backflow prevention pressure to a capillary needle filled with an object to be injected into a cell,
A discharge unit that applies pressure to the capillary needle to discharge the object from the tip of the capillary needle;
A discharge rate calculation unit that calculates a discharge rate that is a discharge amount per unit time of the target object discharged by the discharge unit;
A decompression unit that reduces the pressure applied to the capillary needle by the ejection unit until the ejection rate calculated by the ejection rate calculation unit is equal to or lower than a predetermined threshold;
A microinjection comprising: a backflow prevention pressure determining unit that determines, as the backflow prevention pressure, the pressure when the discharge rate calculated by the discharge rate calculation unit is equal to or less than the predetermined threshold value. apparatus. An imaging device that captures an image of the object ejected by the ejection unit at a predetermined interval time;
The discharge rate calculation unit
The image of the object imaged by the imaging device immediately after the object is discharged by the discharge unit, and after the predetermined interval time has elapsed from immediately after the object is discharged by the discharge unit. The discharge amount of the target object is calculated based on the luminance sum of the difference image from the image of the target object captured by the imaging device, and the calculated discharge amount of the target object is divided by the predetermined interval time. The microinjection apparatus according to claim 1, wherein the ejection rate is calculated by doing so. The decompression unit is
The microinjection device according to claim 1, wherein the pressure applied to the capillary needle by the discharge unit is decreased by a certain amount. An approximate straight line generating unit that generates an approximate straight line of the discharge rate for each pressure applied to the capillary needle by the discharge unit so far;
A zero pressure calculation unit that calculates a zero pressure that is a pressure at which the discharge rate becomes zero on the approximate line generated by the approximate line generation unit;
An average value calculating unit that calculates an average value of the pressure applied this time to the capillary needle by the discharge unit and the zero pressure calculated by the zero pressure calculating unit;
The decompression unit is
The average value calculated by the average value calculation unit is set to a pressure to be applied to the capillary needle next time by the discharge unit, and the pressure applied to the capillary needle by the discharge unit this time is set to the pressure The microinjection apparatus according to claim 1, wherein the microinjection apparatus is reduced to an average value. A backflow prevention pressure measurement method for measuring the backflow prevention pressure in a microinjection device that applies backflow prevention pressure to a capillary needle filled with a target to be injected into a cell,
A discharge step of applying pressure to the capillary needle to discharge the object from the tip of the capillary needle;
A discharge rate calculating step of calculating a discharge rate that is a discharge amount per unit time of the object discharged by the discharge step;
A pressure reduction step of reducing the pressure applied to the capillary needle by the discharge step until the discharge rate calculated by the discharge rate calculation step is equal to or lower than a predetermined threshold;
A backflow prevention pressure determining step for determining, as the backflow prevention pressure, the pressure when the discharge rate calculated by the discharge rate calculation step is equal to or less than the predetermined threshold value. Pressure measurement method.

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