Ultra-microinjection detection and control device based on lensless imaging and method thereof

Abstract

The present invention provides an ultra-microinjection detection and control device based on lensless imaging and a method thereof. A lensless optical liquid level sensor is used to measure a change of a liquid level in an injection needle. A microinjection control unit is used to track the change of the liquid level and correct an injection pressure of the injection pump. Transmitted light generated by a parallel light source passes through a transparent glass tube of the injection needle. Then the transmitted light passes through a light filtering film to reduce the intensity of the parallel light source to a photosensitivity range of a micro linear array image sensor chip. Finally, the transmitted light enters the micro linear array image sensor chip, so that the micro linear array image sensor chip measures the change of the liquid level in the injection needle.

Claims

1. An ultra-microinjection detection and control device based on lensless imaging, wherein the device comprises an injection pump and a needle holder (e); the needle holder (e) is used to hold an injection needle (c); the injection pump is used to provide power needed for injection; the device further comprises a lensless optical liquid level sensor and a microinjection control unit; the lensless optical liquid level sensor is arranged on an outer wall of the injection needle (c), for measuring a change of a liquid level in the injection needle (c); the microinjection control unit is arranged on the needle holder (e), for detecting the change of the liquid level and correcting an injection pressure of the injection pump, so as to stabilize the liquid level in the injection needle (c); the lensless optical liquid level sensor comprises a micro linear array image sensor chip (5), a light filtering film (1), a parallel light source (4) and a holding housing (6); the parallel light source (4) is arranged on an outer side wall of the injection needle (c); the light filtering film (1) is arranged on an outer side wall of the injection needle (c) and located on a transmitted plane of the parallel light source (4); the micro linear array image sensor chip (5) is arranged on an outer surface of the light filtering film (1); the holding housing (6) encloses the micro linear array image sensor chip (5), the light filtering film (1) and the parallel light source (4); transmitted light generated by the parallel light source (4) passes through a transparent glass tube of the injection needle (c); then the transmitted light passes through the light filtering film (1) to reduce the intensity of the parallel light source (4) to a photosensitivity range of the micro linear array image sensor chip (5); finally, the transmitted light enters the micro linear array image sensor chip (5), so that the micro linear array image sensor chip (5) measures the change of the liquid level in the injection needle (c).

2. The ultra-microinjection detection and control device based on lensless imaging according to claim 1, wherein the parallel light source (4) comprises a light-emitting diode (LED) element, an optical fiber and a light homogenizing film; the LED element, the optical fiber and the light homogenizing film are arranged in order from outside to inside.

3. The ultra-microinjection detection and control device based on lensless imaging according to claim 1, wherein the control unit comprises a driver circuit (3) and a flexible circuit board (2); the driver circuit (3) is arranged on the needle holder (e), and the driver circuit (3) is connected to the micro linear array image sensor chip (5) through the flexible circuit board (2); the flexible circuit board (2) is used to acquire measurement information of the micro linear array image sensor chip (5), and correct the injection pressure of the injection pump based on the information; the driver circuit (3) is used to drive the injection pump according to the injection pressure sent by the flexible circuit board (2).

4. The ultra-microinjection detection and control device based on lensless imaging according to claim 1, wherein the micro linear array image sensor chip (5) is an image sensor chip with only one or a few rows of photosensitive units in a y direction and hundreds or thousands of photosensitive units in an x direction.

5. A method for implementing the ultra-microinjection detection and control device based on lensless imaging according to claim 1, wherein the method comprises the following steps: step 1, the light emitted by the parallel light source (4) passes through the transparent glass injection needle (c) and enters the micro linear array image sensor chip (5) via the light filtering film (1); the liquid level in the injection needle (c) presents a virtual image on the micro linear array image sensor chip (5); step 2: the microinjection control unit obtains a minimum injection volume of a liquid according to the virtual image; and step 3: the microinjection control unit corrects a pressure of the injection pump according to the minimum injection volume, so as to stabilize the injection liquid in the injector.

6. The method for implementing the ultra-microinjection detection and control device based on lensless imaging according to claim 5, wherein the minimum injection volume of the liquid is as follows: a theoretical minimum resolution Δx of the micro linear array image sensor chip (5) is expressed as follows: $\begin{array}{cc}\Delta x=\frac{N.Math.a}{\left(P_1-P_0\right){\mathrm{.Math.}}_0},,& \mathrm{Formula}1\end{array}$ wherein, P.sub.0 and P.sub.1 are binary representations of a light intensity in the liquid and in the air; a is a single-pixel length; N is a number of pixels passing from a minimum light intensity P.sub.0 to a maximum light intensity P.sub.1 through a boundary area of the liquid level; according to the theoretical minimum resolution Δx of the micro linear array image sensor chip (5), the minimum injection volume of the liquid is as follows:
V.sub.min=¼.Math.πd.sup.2Δx,  Formula 2 wherein, d is an inner diameter of the injection needle; V.sub.min is a minimum injection volume of the liquid in the injection needle.

7. The method for implementing the ultra-microinjection detection and control device based on lensless imaging according to claim 6, wherein in step 3, the microinjection control unit corrects a pressure of the injection pump according to the following formula: according to an equation for a liquid level balance in the injection needle (c):
F.sub.b=F.sub.c+F.sub.G+F.sub.a,  Formula 3 a balance pressure F.sub.b of the injection pump is obtained, wherein, F.sub.c is a capillary force generated by a needle end to the liquid in the injection needle; F.sub.G is a vertical component of a gravity of the liquid in the injection needle during injection; F.sub.a is an ambient pressure; the liquid in the injection needle (c) has a capillary force at the needle tip and the needle end; in an actual application, the needle tip will be immersed in water or a liquid mixed with water, so that the capillary force at the needle tip disappears; therefore, only the capillary force F.sub.c generated by the needle end to the liquid in the injection needle remains; the capillary force F.sub.c at a boundary is:
F.sub.c=2πrσ.sub.A cos θ,  Formula 4 the vertical component F.sub.G of the gravity of the liquid in the injection needle during injection is:
F.sub.G=πr.sup.2ρgh.Math.sin β,  Formula 5 wherein, σ.sub.A cos θ is a tensile force acting on a water column on a three-phase perimeter of a unit length within the capillary tube, measured by an experiment; θ represents an angle between a concave surface of the liquid in the glass capillary tube and a wall of the glass tube; r is an inner radius of the injection needle; β is an angle between the injection needle and a horizontal plane during an actual injection; the microinjection control unit corrects the balance pressure of the injection pump according to the minimum injection volume, so as to stabilize the injection liquid in the injector.

8. The method for implementing the ultra-microinjection detection and control device based on lensless imaging according to claim 7, wherein the stability of the injection liquid in the injector refers to a balance between the injection pressure and a liquid flow of the injection needle; the injection pressure and the liquid flow satisfy: during the injection, a relationship between an injection pressure and a flow of the needle tip is calculated by using micro-hydrodynamics; the flow Q of the needle tip is estimated by a Poisson equation: $\begin{array}{cc}Q=\frac{d^{4}p}{128\mu L},& \mathrm{Formula}6\end{array}$ wherein, p is an injection pressure, μ is a dynamic viscosity, and L is a length of a tube; the needle tip is simplified to a cross section of a tube with a uniformly varying cross section, d=2 tan θ; the cross section is derived, and the flow in a tapered tube satisfies $\frac{\mathrm{dp}}{\mathrm{dx}}=\mathrm{const}\theta ;$ the relationship between the pressure and the flow is: $\begin{array}{cc}{\int }_{d_1}^{d_2}\frac{64\mathrm{Qu}}{\pi \tan \alpha d^4}d\left(d\right),& \mathrm{Formula}7\end{array}$ wherein, d.sub.1 and d.sub.2 are outlet and inlet diameters of the needle tip, respectively; α is an included angle of the needle tip.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic structural diagram of a conventional microinjection system, where reference numeral a represents a petri dish; reference numeral b represents a microscope; reference numeral c represents an injection needle; reference numeral d represents a robotic arm; reference numeral e represents a needle holder; reference numeral f represents an injection pump, which can be implemented by a pneumatic microinjection pump;

(2) FIG. 2 is a detail drawing of an injection needle in FIG. 1, where reference numeral g represents a detail drawing of a needle tip; reference numeral h represents an outer diameter of the needle tip, which is 20 μm; reference numeral i represents an outer diameter of a needle tube, which is 1,000 μm;

(3) FIG. 3 is a schematic structural diagram of an ultra-microinjection detection and control device based on lensless imaging according to Specific Implementation 1;

(4) FIG. 4 is an exploded view of a lensless optical liquid level sensor in FIG. 3;

(5) FIG. 5 is a schematic diagram showing a relationship between a light intensity collected by a micro linear array image sensor chip and displacement;

(6) FIG. 6 is a schematic diagram showing a capillary force of a liquid level in an injection needle; and

(7) FIG. 7 is a needle tip fluid model of an injection needle.

DETAILED DESCRIPTION

(8) Specific Implementation 1: This implementation is described in detail with reference to FIG. 3 and FIG. 4. This implementation provides an ultra-microinjection detection and control device based on lensless imaging, where the device includes an injection pump and a needle holder e; the needle holder e is used to hold an injection needle c; the injection pump is used to provide pressure needed for injection; the device further includes a lensless optical liquid level sensor and a microinjection control unit.

(9) The lensless optical liquid level sensor is arranged on an outer wall of the injection needle c, for measuring a change of a liquid level in the injection needle c.

(10) The microinjection control unit is arranged on the needle holder e, for tracking a change of the liquid level and correcting an injection pressure of the injection pump, so as to stabilize the liquid level in the injection needle c.

(11) The lensless optical liquid level sensor includes a micro linear array image sensor chip 5, a light filtering film 1, a parallel light source 4 and a holding housing 6.

(12) The parallel light source 4 is arranged on an outer side wall of the injection needle c. The light filtering film 1 is arranged on an outer side wall of the injection needle c and located on a transmitted plane of the parallel light source 4. The micro linear array image sensor chip 5 is arranged on an outer surface of the light filtering film 1. The holding housing 6 encloses the micro linear array image sensor chip 5, the light filtering film 1 and the parallel light source 4.

(13) Transmitted light generated by the parallel light source 4 passes through a transparent glass tube of the injection needle c. Then the transmitted light passes through the light filtering film 1 to reduce the intensity of the parallel light source 4 to a photosensitivity range of the micro linear array image sensor chip 5. Finally, the transmitted light enters the micro linear array image sensor chip 5, so that the micro linear array image sensor chip 5 measures the change of the liquid level in the injection needle c.

(14) In this implementation, the micro linear array image sensor chip 5 is held on the glass injection needle. The micro linear array image sensor chip 5 is connected to the microinjection control unit through a flexible circuit board. The microinjection control unit can be arranged on the needle holder and a micromanipulator, etc.

(15) The light filtering film can reduce an incident light intensity in a specific spectral range at a specific ratio. The function of the light filtering film is to eliminate the interference of external stray light on the sensor, and reduce the intensity of the parallel light source to the photosensitivity range of the image sensor.

(16) The parallel light source is composed of a light-emitting diode (LED) element, an optical fiber and a light homogenizing film, providing homogeneous and stable transmitted light.

(17) The holding housing is a structure integrating the micro linear array image sensor chip, the light filtering film and the parallel light source, and creating a closed environment to reduce the entry of external light. The holding housing also has a rubber structure for providing resistance to ensure stable measurement when held on the glass needle. The above structure is shown in FIG. 4.

(18) The control unit mainly completes an image acquisition and processing algorithm, a flow control algorithm and a communication program, etc. The hardware can be assumed by computing chips such as a single-chip computer, a digital signal processor DSP and a field programmable gate array FPGA.

EMBODIMENT 1

(19) In the present invention, the micro linear array image sensor chip may use an S10226 linear complementary metal-oxide-semiconductor transistor (CMOS) image sensor produced by Hamamatsu as a sensor chip. This sensor chip has 12 sensing bits. In a photosensitive area, a single pixel has a size of 7×125 μm, and a linear array has a total of 1,024 pixels. The photosensitive area has a total length of 8 mm and overall dimensions of 9.1 mm×2.4 mm×1.6 mm. A surface of the sensor is covered with a 90% light filtering film. The sensor is provided with an external light source composed of a LED element and an optical fiber, with a light intensity of about 150 lux. The glass injection needle can be pulled from a glass capillary tube with an outer diameter of 1 mm and an inner diameter of 0.5 mm. The pneumatic microinjection pump can be used with a FemtoJet 4i picoliter upgrade microinjection pump produced by Eppendorf.

(20) In this embodiment, it is assumed that the light passing through the liquid level can saturate the sensor pixels, the light passing through the air does not reach a threshold of the sensor after passing through the light filtering film, and a boundary of the liquid level has a light intensity difference of 4,096. An electrical noise fluctuation, which is set to 10%, is substituted into Formula 1 to obtain a theoretical minimum liquid level resolution as follows:

(21) $\begin{array}{cc}\Delta x=\frac{N.Math.a}{\left(P_1-P_0\right){\mathrm{.Math.}}_0}=\frac{7\mu m}{4096\times 10\%}=0.017\mu m,& \mathrm{Formula}1\end{array}$
Δx is substituted into Formula 2:

(22) $\begin{array}{cc}{V}_{\min }=\frac{1}{4}.Math.\pi d^2\Delta x,& \mathrm{Formula}2\end{array}$

(23) The minimum injection volume is calculated to be 3.4 pL. The resolution exceeds the requirements of the minimum injection volume of the microinjection pump and an injection volume of a general life science experiment. Therefore, the resolution meets the need of actual use.

(24) For an injection needle having an inner diameter of 0.5 mm, the boundary of the liquid level is about 35 μm in width. Therefore, when the S10226 sensor is used, a maximum sensing range of the sensor in terms of liquid level is (8 mm−2×0.035 mm)=7.93 mm, which is equal to a maximum range of 1.56 μL in terms of liquid volume. By calculating based on a single-injection volume of 2 nL in a general experiment, the volume of the liquid filled once in the present application can be used for 780 injections, which meets a continuous injection requirement of most experiments.

(25) Specific Implementation 2: This implementation further describes the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 1. In this implementation, the parallel light source 4 includes a LED element, an optical fiber and a light homogenizing film. The LED element, the optical fiber and the light homogenizing film are arranged in order from outside to inside.

(26) Specific Implementation 3: This implementation further describes the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 1. In this implementation, the control unit includes a driver circuit 3 and a flexible circuit board 2. The driver circuit 3 is arranged on the needle holder e, and the driver circuit 3 is connected to the micro linear array image sensor chip 5 through the flexible circuit board 2. The flexible circuit board 2 is used to acquire measurement information of the micro linear array image sensor chip 5, and correct the injection pressure of the injection pump based on the information. The driver circuit 3 is used to drive the injection pump according to the injection pressure sent by the flexible circuit board 2.

(27) Specific Implementation 4: This implementation further describes the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 1. In this implementation, the micro linear array image sensor chip 5 is an image sensor chip with only one or a few rows of photosensitive units in a y direction and hundreds or thousands of photosensitive units in an x direction.

(28) In this implementation, the linear array image sensor chip is an image sensor chip with only one or a few rows of photosensitive units in a y direction and hundreds or thousands of photosensitive units in an x direction. A length of the sensor chip in the x direction determines the maximum range of liquid level measurement. A size of the photosensitive unit in the x direction is directly proportional to a measurement accuracy of the sensor. A size of the photosensitive unit in the y direction is inversely proportional to a need of the sensor for the brightness of the light source. The sensitivity of the photosensitive unit to light intensity is within a certain range, and the measured light intensity must be within this range.

(29) Specific Implementation 5: A method for implementing the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 1, where the method includes the following steps: step 1, the light emitted by the parallel light source 4 passes through the transparent glass injection needle c and enters the micro linear array image sensor chip 5 via the light filtering film 1; the liquid level in the injection needle c presents a virtual image on the micro linear array image sensor chip 5; step 2: the microinjection control unit obtains a minimum injection volume of the liquid according to the virtual image; and step 3: the microinjection control unit corrects a pressure of the injection pump according to the minimum injection volume, so as to stabilize the injection liquid in the injector.

(30) Specific Implementation 6: This implementation further describes the method for implementing the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 5. In this implementation, the minimum injection volume of the liquid is:

(31) a theoretical minimum resolution Δx of the micro linear array image sensor chip 5 is expressed as follows:

(32) $\begin{array}{cc}{\Delta }_x=\frac{N.Math.a}{\left(p_1-p_0\right){\mathrm{.Math.}}_0},,& \mathrm{Formula}1\end{array}$
where, P.sub.0 and P.sub.1 are binary representations of a light intensity in the liquid and in the air; a is a single-pixel length; N is a number of pixels passing from a minimum light intensity P.sub.0 to a maximum light intensity P.sub.1 through a boundary area of the liquid level; according to the theoretical minimum resolution Δx of the micro linear array image sensor chip 5, the minimum injection volume of the liquid is as follows:
V.sub.min=¼.Math.πd.sup.2Δx,  Formula 2
where, d is an inner diameter of the injection needle; V.sub.min is a minimum injection volume of the liquid in the injection needle.

(33) In this implementation, a signal acquired by the micro linear array image sensor chip includes a noise fluctuation of the light source and the sensor. Therefore, the light intensity resolution represented by (P.sub.1−P.sub.0) needs to be multiplied by an error coefficient ε.sub.0, and the value needs to be determined based on a noise width of the sensor.

(34) Specific Implementation 7: This implementation further describes the method for implementing the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 6. In this implementation, in step 3, the microinjection control unit corrects a pressure of the injection pump according to the following formula:

(35) according to an equation for a liquid level balance in the injection needle c:
F.sub.b=F.sub.c+F.sub.G+F.sub.a,  Formula 3
a balance pressure F.sub.b of the injection pump is obtained;
where, F.sub.c is a capillary force generated by a needle end to the liquid in the injection needle;
F.sub.G is a vertical component of a gravity of the liquid in the injection needle during injection;
F.sub.a is an ambient pressure.

(36) The liquid in the injection needle c has a capillary force at the needle tip and the needle end. In an actual application, the needle tip will be immersed in water or a liquid mixed with water, so that the capillary force at the needle tip disappears. Therefore, only the capillary force F.sub.c generated by the needle end to the liquid in the injection needle remains. The capillary force F.sub.c at the boundary is:
F.sub.c=2πrσ.sub.A cos θ,  Formula 4
the vertical component F.sub.G of the gravity of the liquid in the injection needle during injection is:
F.sub.G=πr.sup.2ρgh.Math.sin β,  Formula 5

(37) where, σ.sub.A cos θ is a tensile force acting on a water column on a three-phase perimeter of a unit length within the capillary tube, measured by an experiment; θ represents an angle between a concave surface of the liquid in the glass capillary tube and a wall of the glass tube; r is an inner radius of the injection needle; β is an angle between the injection needle and a horizontal plane during an actual injection.

(38) The microinjection control unit corrects the balance pressure of the injection pump according to the minimum injection volume, so as to stabilize the injection liquid in the injector.

(39) Specific Implementation 8: This implementation further describes the method for implementing the ultra-microinjection detection and control device based on lensless imaging as described in Specific Implementation 7. In this implementation, the stability of the injection liquid in the injector refers to a balance between the injection pressure and a liquid flow of the injection needle.

(40) The injection pressure and the liquid flow satisfy a relationship as follows.

(41) During the injection, a relationship between an injection pressure and a flow of the needle tip is calculated by using micro-hydrodynamics. The flow Q of the needle tip is estimated by a Poisson equation:

(42) $\begin{array}{cc}Q=\frac{d^{4}p}{128\mu L},,& \mathrm{Formula}7\end{array}$
where, p is an injection pressure, μ is a dynamic viscosity, and L is a length of a tube.

(43) The needle tip is simplified to a cross section of a tube with a uniformly varying cross section, d=2 tan θ. The cross section is derived, and the flow in a tapered tube satisfies

(44) $\frac{\mathrm{dp}}{\mathrm{dx}}=\mathrm{const}\theta .$
The relationship between the pressure and the flow is:

(45) $\begin{array}{cc}{\int }_{d_1}^{d_2}\frac{64\mathrm{Qu}}{\pi \tan \alpha d^4}d\left(d\right),,& \mathrm{Formula}8\end{array}$
where, d.sub.1 and d.sub.2 are outlet and inlet diameters of the needle tip, respectively; α is an included angle of the needle tip.

(46) From the above derivation, the corresponding injection pressure and injection time can be calculated. When the control unit controls the injection pump to perform the injection operation, the control unit tracks the change of the liquid level to monitor a change in the injection volume. In this way, the control unit corrects the injection parameters and prompts the occurrence of common faults such as blockage.