Micro-droplet comparison calibration device and dynamic comparison calibration method

Abstract

The invention discloses a liquid drop comparison and calibration method / device, and belongs to the technical field of ultrasonic pipetting. According to the comparison calibration method, the standard steel ball is bounced to the air to simulate the process that a micro-droplet is excited to the air, an industrial camera is adopted to shoot a flight image of the standard spherical steel ball, the contour of the standard steel ball is recognized, and the size of the steel ball is calculated based on the constructed rotational symmetry model of the steel ball. The measurement result is compared with the standard value of the standard steel ball, and the micro-droplet volume measurement error combined with the industrial camera is evaluated.

Description

Technical Field

[0001] The invention relates to the technical field of ultrasonic liquid transfer, and in particular to a micro-droplet comparison calibration device and a dynamic comparison calibration method. Background Art

[0002] Acoustic Droplet Ejection (ADE) technology has made significant progress in recent years. This technology uses focused ultrasound to precisely control the ejection of droplets, breaking through the limitations of traditional liquid transfer tools. By adjusting the frequency and intensity of ultrasound, the number and volume of droplets can be precisely controlled. If ultrasonic pipetting technology is to be applied in fields such as biotechnology, drug development and in vitro diagnostics, the required liquid volume will be reduced from milliliters and microliters to nanoliters or picoliters of droplets.

[0003] In the existing ultrasonic pipetting technology, the mainstream method for measuring the volume of micro-droplets during ultrasonic pipetting is the droplet volume observation method combined with a high-speed industrial camera. This method uses a high-speed industrial camera to capture the process of droplet falling, extracts the droplet morphology information from the captured droplet image, and combines it with the droplet volume solution model to calculate the droplet volume.

[0004] However, when the droplets are stimulated to fly into the air, their flight trajectory does not rise or fall vertically, but rather moves in a similar manner to a horizontal projection. The horizontal distance between the droplets and the camera will change, while the focal length of the camera remains unchanged when the industrial camera is used for shooting. This will lead to certain errors, and the accuracy of calculating the droplet volume depends on the accuracy of the droplet volume model.

[0005] To obtain the error size of the droplet volume observation method of industrial cameras, the more mainstream method is to use the droplet weighing method to collect and weigh a known number of droplets to measure the average droplet volume, and then compare the weighed droplet volume with the volume value calculated by the industrial camera droplet volume observation method to obtain the error size of this method. However, the disadvantage of this method is that the error may be large considering the evaporation loss of droplets, and the experimental conditions are harsh. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a micro-droplet comparison calibration device and a dynamic comparison calibration method, aiming to solve the technical problems in the prior art of high cost of droplet volume calibration equipment and low accuracy of calibration results.

[0007] To achieve the above object, the present invention provides the following technical solution: a micro-droplet comparison and calibration device, characterized in that it includes:

[0008] A base, used to provide a mounting position for each component;

[0009] Standard steel balls, used as standard reference for comparative calibration;

[0010] A piston, mounted on a base, for storing a standard steel ball;

[0011] A spring is installed in the piston. A push rod is fixed at one end of the spring. The push rod abuts against the standard steel ball and is used to utilize the elastic potential energy of the spring to eject the standard steel ball from the piston.

[0012] The trigger can be turned over and installed on the piston, and one end of the trigger extends into the piston to abut against the ejection rod, which is used to clamp or release the spring, so that the spring continues to bounce the standard steel ball;

[0013] An industrial camera, mounted near the base, is used to capture images of a simulated droplet flight process when a standard steel ball is bounced;

[0014] The host computer is connected to the industrial camera for receiving images taken by the industrial camera, preprocessing the taken standard steel ball flight images, and calculating the volume parameters of the steel ball using a preset software program and recording them in a table;

[0015] The parallel light source is installed at a position opposite to the industrial camera and is used to provide light for the industrial camera.

[0016] As a further improvement of the present invention, a Z-direction dial is also installed on the base, and an arc-shaped adjustment groove is opened on the Z-direction dial. One end of the piston is hinged to the base so that the piston can be flipped up and down. The side wall of the piston is threadedly connected with an adjusting nut, and the adjusting nut is inserted into the adjustment groove to cooperate with the Z-direction dial to position the flipping angle of the piston to determine the angle between the piston and the Z direction.

[0017] As a further improvement of the present invention, a base frame is also provided below the base, and an annular angle scale line is engraved on the upper side of the base frame. A rotating pillar is rotatably provided on the upper side of the base frame at the center of the annular angle scale line. The base is installed at the upper end of the rotating pillar and rotates around the rotating pillar as the axis. A base pointer is also fixed to the lower side of the base for pointing to the annular angle scale line, thereby determining the angle between the piston and the X direction.

[0018] Another aspect of the present invention provides a comparative calibration method, comprising the following steps:

[0019] Step 1: Adjust the adjusting nut and the rotating support to make the piston face a certain angle, and record the angle between the piston and the Z direction and the X direction;

[0020] Step 2: Pull the trigger to make the standard steel ball fly into the air by the spring;

[0021] Step 3: Use an industrial camera to capture a flight image containing a standard steel ball, and feed the captured flight image back to the host computer;

[0022] Step 4: Use the software program built into the host computer to pre-process the acquired flight image of the standard steel ball, calculate the volume of each frame of the standard steel ball flight image, and record the volume data of each standard steel ball in a table;

[0023] Step 5: Compare the known volume of the standard steel ball with the measurement result of the standard steel ball calculated by image preprocessing to evaluate the error of the droplet volume measurement combined with the industrial camera.

[0024] As a further improvement of the comparative calibration method, the specific method for calculating the volume of each frame of the standard steel ball (3) flight image in step 4 is: image preprocessing is performed on the standard steel ball (3) flight image to obtain the outline of the steel ball in the standard steel ball (3) flight image, and the volume of the standard steel ball (3) is calculated by constructing a three-dimensional rotation model of the steel ball in combination with the actual size of the standard steel ball (3) flight image.

[0025] As a further improvement of the comparative calibration method, the specific steps of constructing the three-dimensional rotation model of the steel ball are as follows:

[0026] Step 41: Take the standard size camera calibration plate as the object, shoot the calibration plate image with the industrial camera, and obtain the actual size of the image captured as W×H; establish the calibration relationship between camera pixels and actual size, and calculate the thickness of one pixel unit of the droplet image according to the following formula:

[0027] d=H / D pi

[0028] Where H is the height of the actual size of the image; d is the unit thickness of the pixel; D pi is the vertical resolution of the industrial camera;

[0029] Step 42: Obtain the calibration relationship between camera pixels and actual size. The volume calculation of the standard steel ball 3 is based on the rotational symmetry model. The volume V of the standard steel ball 3 is calculated according to the following formula:

[0030]

[0031] Among them, D i is the actual diameter of the standard steel ball (3) corresponding to each row of pixels.

[0032] As a further improvement of the comparative calibration method, the actual diameter D of the standard steel ball (3) corresponding to each row of pixels is i It is calculated by the following formula:

[0033] Di =d(u max -u min ) i

[0034] Among them, u max and u min is the edge position of the pixel detected along the row direction of the standard steel ball 3 image.

[0035] The beneficial effects of the present invention include using a standard steel ball to be bounced into the air to simulate the process of micro-droplets being excited into the air, using an industrial camera to capture the flight image of the standard spherical steel ball, identifying the outline of the standard steel ball, calculating the volume of the steel ball based on constructing a rotationally symmetric model of the steel ball, comparing the measurement results with the standard value of the standard steel ball, and evaluating the error size of the micro-droplet volume measurement combined with the industrial camera.

[0036] This comparative calibration method uses a high-speed camera to capture a flying image of a known standard spherical steel ball, identifies the outline of the standard steel ball, calculates the droplet volume based on a rotationally symmetric model of the droplet, and compares the calculated result with the standard volume value of the standard steel ball. This can evaluate the size of the droplet volume measurement error combined with an industrial camera. There is no need to use the droplet weighing method in the existing mainstream technology, and there is no need to consider the evaporation of micro-droplets during calibration. The operation is simple and convenient, which effectively reduces the calibration cost and improves the calibration accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of a droplet comparison calibration device according to an embodiment of the present application;

[0038] Figure 2 is a schematic diagram of actual droplet flight in an embodiment of the present application;

[0039] Figure 3 This is a schematic diagram of obtaining a standard steel ball flight image in an embodiment of the present application.

[0040] Figure 4 It is a schematic diagram of a camera calibration plate for camera photography according to an embodiment of the present application.

[0041] Figure 5 Schematic diagram of the rotational symmetry model used in calculating the volume of the steel ball in the embodiment of the present application

[0042] Figure 6 Schematic diagram of the width of each row of pixels in the standard steel ball 3 image of the embodiment of the present application DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with the embodiments shown in the accompanying drawings, wherein the X direction and the Z direction mentioned in this embodiment represent the horizontal direction and the vertical direction.

[0044] Reference Figure 1 As shown, this embodiment first provides a micro-droplet comparison calibration device, which specifically includes:

[0045] Base, used to install various components;

[0046] Standard steel ball 3, used as a standard reference for comparative calibration;

[0047] A piston 5, used for storing a standard steel ball 3;

[0048] The spring 6 uses the elastic potential energy of the spring 6 to eject the standard steel ball 3 from the piston 5;

[0049] The trigger 7 is used to lock or release the spring 6 to eject the standard steel ball;

[0050] The adjusting nut 8 is used to adjust the launching angle of the standard steel ball 3 in the vertical direction;

[0051] A Z-direction scale plate 9 is engraved with angle scale lines corresponding to the Z-direction. An adjusting nut 8 is located on the Z-direction scale plate 9. The angle between the piston 5 and the Z-direction can be determined by adjusting the nut 8 at the position of the Z-direction scale plate 9.

[0052] The base pointer 10 is symmetrically fixed to the bottom of the base and rotates around the Z direction together with the base;

[0053] A rotating support 11 is disposed at the bottom of the base, so that the base can rotate around the Z direction;

[0054] The base frame 12 is used to bear the weight of the base and its upper components, and an annular angle scale is engraved on its upper surface. When the base rotates around the Z axis through the rotating support 11, the angle between the piston 5 and the X direction can be determined by the position of the annular angle scale on the upper surface of the base frame 12 by the base pointer 10;

[0055] Industrial camera 2 is used to capture images of the standard steel ball 3 simulating the flight process of the droplet; the industrial camera 2 is a high-resolution black-and-white CCD camera with a resolution of 1280×1024 pixels. The image frame rate captured by the CCD camera is 1000 frames per second, and it is equipped with a camera multiplier. The magnification of the camera multiplier can be adjusted to image objects of nanoliter size. In practical applications, the magnification of the camera multiplier can be adjusted according to the parameters of the standard steel ball 3;

[0056] The host computer 1 is used to receive the flying image of the standard steel ball 3 taken by the industrial camera 2, pre-process the flying image of the standard steel ball 3, calculate the volume parameters of the steel ball using a preset software program and record them in a table;

[0057] Parallel light source 4, in order to ensure the clarity of the field of view of the image taken by the industrial camera 2, the parallel light source is facing the industrial camera, and the intensity of the parallel light source is adjustable from 0 to 48W;

[0058] In a preferred embodiment, in order to better simulate the real micro-droplet size, the diameters of the standard steel balls used are 0.3mm, 0.4mm, and 0.5mm. The corresponding micro-droplet volumes are 14.14nl, 33.51nl, and 65.45nl;

[0059] In a preferred embodiment, to better simulate real micro-droplets (such as Figure 2 As shown in the figure, during the flight process in the air, the rotatable angle of the adjustment nut 8 and the Z direction angle are adjustable from 0° to 90°, and the rotation angle of the rotating support 11 around the Z axis is adjustable from 0° to 180°.

[0060] On the other hand, this embodiment provides a dynamic comparison calibration method, comprising the following steps:

[0061] Adjust the adjusting nut 8 and the rotating support 11 to make the piston 5 face a certain angle, and record the angle between the direction of the piston 5 and the Z direction and the X direction;

[0062] Pull the trigger 7, so that the standard steel ball 3 is ejected into the air by the spring 6;

[0063] An industrial camera 2 is used to capture an image containing a standard steel ball 3 in flight, and the captured image of the standard steel ball 3 in flight (such as Figure 3 Feedback to the host computer;

[0064] The acquired flying image of the standard steel ball 3 is preprocessed by using the software program of the host computer 1, the volume of each frame of the flying image of the standard steel ball 3 is calculated, and the volume data of each standard steel ball 3 is recorded in a table.

[0065] Furthermore, the steps of calculating the volume of each frame of the standard steel ball 3 flight image include:

[0066] Performing image preprocessing on the standard steel ball 3 flight image to obtain the outline of the steel ball in the standard steel ball 3 flight image, and calculating the volume of the standard steel ball 3 by constructing a three-dimensional rotation model of the steel ball in combination with the actual size of the standard steel ball 3 flight image;

[0067] like Figure 4 As shown in the figure, a standard-sized camera calibration plate is used as the object, and the industrial camera is used to take 6 images of the calibration plate, and the actual size of the image is obtained as W×H; the calibration relationship between the camera pixel and the actual size is established, and the thickness of one pixel unit of the droplet image is calculated according to the following formula:

[0068] d=H / D pi

[0069] Where H is the height of the actual size of the image;

[0070] d is the unit thickness of one pixel;

[0071] D pi is the vertical resolution of the industrial camera;

[0072] like Figure 5 As shown, when the calibration relationship between the camera pixel and the actual size is obtained, the volume calculation of the standard steel ball 3 is based on the rotational symmetry model, and the volume V of the standard steel ball 3 is calculated according to the following formula:

[0073]

[0074] Di is the actual diameter of the standard steel ball 3 corresponding to each row of pixels, which can be obtained according to the following formula:

[0075] D i =d(u max -u min ) i

[0076] like Figure 6 Shown max and u min is the edge position of the pixel detected along the row direction of the standard steel ball 3 image;

[0077] The known volume of the standard steel ball 3 is compared with the volume measurement result of the standard steel ball 3 calculated by image preprocessing to evaluate the error of the droplet volume measurement combined with the industrial camera.

[0078] The comparative calibration method uses a high-speed camera to capture a flying image of a known standard spherical steel ball, identifies the outline of the standard steel ball 3, calculates the droplet volume based on a rotationally symmetric model of the droplet, and compares the calculated result with the standard volume value of the standard steel ball 3. This can evaluate the size of the droplet volume measurement error combined with an industrial camera without using the droplet weighing method in the existing mainstream technology, and without considering the evaporation of micro-droplets during calibration. The operation is simple and convenient, which effectively reduces the calibration cost and improves the calibration accuracy.

[0079] This embodiment provides the following practical operation process:

[0080] 1. Place a standard steel ball 3 to be shot in the piston 5, and press down the spring 6 so that it is stuck by the trigger 7;

[0081] 2. Manually adjust the rotation angle of the adjustment nut 8 and the rotating support 11 to make the piston 5 face the expected angle, and record the angle between the piston 5 and the Z direction and the X direction;

[0082] 3. Turn on the industrial camera 2 in advance, put the industrial camera 2 in the recording mode, pull the trigger 7, make the standard steel ball 3 fly, and the industrial camera 2 will obtain the flying image of the standard steel ball 3;

[0083] 4. After the video recording is completed, the industrial camera 2 is turned off, and the host computer 1 is used to automatically export and filter the video recording, obtain the flight image with the standard steel ball 3, perform image preprocessing on the image, calculate the parameters of the standard steel ball 3, and record the results in a table;

[0084] 5. Compare the known volume of the standard steel ball 3 calculated by image preprocessing to the measurement result of the standard steel ball 3, so as to evaluate the error of the droplet volume measurement combined with the industrial camera.

[0085] Furthermore, in this embodiment, the above-mentioned micro-droplet comparison calibration device and dynamic comparison calibration method are used to conduct experiments, and a standard steel ball 3 with a diameter of 0.4 mm is used as the experimental object. Since the deviation angle of the droplet from the Z direction will not be too large under normal circumstances, the actual flight situation of the droplet is simulated, and the rotation angle of the adjustment nut 8 and the Z direction angle are set to 0°, 10°, 20°, and 30° respectively, and the rotation angle of the rotating support 11 around the Z axis is set to 0°, 45°, 90°, 135°, and 180° respectively for experiments. The results are shown in Table 1.

[0086] Table 1 records the error rate of a set of standard steel balls simulating droplet flight calculations

[0087]

[0088]

[0089] In summary, the comparative calibration device and comparative calibration method of this embodiment use the known volume of the standard steel ball 3 to compare the measurement result of the standard steel ball 3 calculated by image preprocessing, so as to evaluate the size of the droplet volume measurement error combined with the industrial camera. There is no need to adopt the droplet weighing method in the existing mainstream technology, and there is no need to consider the evaporation of micro-droplets during calibration. The operation is simple and convenient, which effectively reduces the calibration cost and improves the calibration accuracy.

[0090] The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A micro-droplet comparison calibration device, characterized in that: include: A base, used to provide a mounting position for each component; Standard steel balls (3), used as standard reference objects for comparative calibration; A piston (5) is mounted on the base and is used to store the standard steel ball (3); A spring (6), the spring (6) being installed in the piston (5), a push rod being fixed at one end of the spring (6), the push rod being in contact with the standard steel ball (3), and being used to utilize the elastic potential energy of the spring (6) to eject the standard steel ball (3) from the piston (5); A trigger (7) which can be turned over and mounted on the piston (5), and one end of which extends into the piston (5) to abut against the ejection rod, and is used to clamp or release the spring, so that the spring continues to exert its elastic force to bounce the standard steel ball (3); An industrial camera (2) is installed near the base and is used to capture images of the simulated droplet flight process when the standard steel ball (3) is bounced; The host computer (1) is connected to the industrial camera (2) for receiving images captured by the industrial camera (2), pre-processing the captured images of the standard steel ball (3) in flight, and calculating the volume parameters of the steel ball using a preset software program and recording them in a table; A parallel light source (4) is installed at a position opposite to the industrial camera (2) and is used to provide light to the industrial camera (2).

2. The micro-droplet comparison and calibration device according to claim 1, characterized in that: A Z-direction scale plate (9) is also mounted on the base, and an arc-shaped adjustment groove is provided on the Z-direction scale plate (9). One end of the piston (5) is hinged to the base so that the piston (5) can be turned upside down. An adjustment nut (8) is threadedly connected to the side wall of the piston (5). The adjustment nut (8) is inserted into the adjustment groove to cooperate with the Z-direction scale plate (9) to position the turning angle of the piston (5) so as to determine the angle between the piston (5) and the Z direction.

3. The micro-droplet comparison and calibration device according to claim 2, characterized in that: A base frame (12) is also provided below the base, and an annular angle scale line is engraved on the upper side of the base frame (12). A rotating support (11) is rotatably provided on the upper side of the base frame (12) at the center of the annular angle scale line. The base is mounted on the upper end of the rotating support (11) and rotates around the rotating support (11) as an axis. A base pointer (10) is also fixed on the lower side of the base for pointing to the annular angle scale line, thereby determining the angle between the piston (5) and the X direction.

4. A dynamic comparison calibration method, applied to the comparison calibration device according to any one of claims 1 to 3, characterized in that: The steps include: Step 1, adjusting the adjusting nut (8) and the rotating support (11) to make the piston (5) face a certain angle, and recording the angle between the direction of the piston (5) and the Z direction and the X direction; Step 2: Pull the trigger (7) to make the standard steel ball (3) be ejected into the air by the spring (6); Step 3, using an industrial camera (2) to capture a flight image containing the standard steel ball (3), and feeding the captured flight image back to the host computer (1); Step 4, using the software program built into the host computer (1) to perform image preprocessing on the acquired flight image of the standard steel ball (3), calculating the volume of each frame of the flight image of the standard steel ball (3), and recording the volume data of each standard steel ball (3) in a table; Step 5, using the known volume of the standard steel ball (3) to compare the volume measurement result of the standard steel ball (3) calculated by image preprocessing, so as to evaluate the error size of the droplet volume measurement combined with the industrial camera.

5. The dynamic comparison calibration method according to claim 4, characterized in that: The specific method for calculating the volume of each frame of the standard steel ball (3) flight image in step 4 is: performing image preprocessing on the standard steel ball (3) flight image to obtain the outline of the steel ball in the standard steel ball (3) flight image, and calculating the volume of the standard steel ball (3) by constructing a three-dimensional rotation model of the steel ball in combination with the actual size of the standard steel ball (3) flight image.

6. The dynamic comparison calibration method according to claim 4, characterized in that: The specific steps of constructing the three-dimensional rotation model of the steel ball are as follows: Step 41: Take the standard size camera calibration plate as the object, shoot the calibration plate image with the industrial camera, and obtain the actual size of the image captured as W×H; establish the calibration relationship between camera pixels and actual size, and calculate the thickness of one pixel unit of the droplet image according to the following formula: d=H / D pi Where H is the height of the actual size of the image; d is the unit thickness of the pixel; D pi is the vertical resolution of the industrial camera; Step 42: Obtain the calibration relationship between camera pixels and actual size. The volume calculation of the standard steel ball 3 is based on the rotational symmetry model. The volume V of the standard steel ball 3 is calculated according to the following formula: Among them, D i is the actual diameter of the standard steel ball (3) corresponding to each row of pixels.

7. The dynamic comparison calibration method according to claim 5, characterized in that: The actual diameter D of the standard steel ball (3) corresponding to each row of pixels i It is calculated by the following formula: D i =d(u max -u min ) i Among them, u max and u min is the edge position of the pixel detected along the row direction of the standard steel ball 3 image.

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