CN110653016B - Pipetting system and calibration method thereof - Google Patents
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Abstract
The disclosure relates to a pipetting system and a calibration method thereof, belongs to the technical field of pipetting equipment, and is used for solving the calibration problem of the pipetting equipment. The calibration method of the pipetting system comprises the following steps: capturing a first image of the calibration point through an image capturing device; obtaining a first offset of a central point of the image capturing device relative to a calibration point in the first image, and calibrating the image capturing device according to the first offset; capturing a second image of the tip of a pipette of the pipetting system and a third image after the tip is displaced by the calibrated image capturing device; obtaining a second offset of the third image relative to the tip of the second image; obtaining the focus distance offset of the definition value of the third image relative to the preset definition value according to the displacement, the variation of the definition value of the third image relative to the second image and a preset mapping relation table of the focus distance and the definition value; calibrating the pipette in accordance with the second offset and the focus distance offset. The present disclosure enables calibration of pipetting systems.
Description
Technical Field
The disclosure relates to the technical field of pipetting equipment, in particular to a pipetting system and a calibration method thereof.
Background
The extraction and injection of liquids is a procedure that is often required in industrial applications. In the automatic process, the liquid can be extracted and injected by using the liquid-transferring equipment, and the operation requirement of micro-liquid transferring is met by the liquid-transferring pipe and the rotating disc of the liquid-transferring equipment.
In practical application, different pipettes are required to be used for different liquids and different operation scenes. In the process of assembling and disassembling the pipette, due to assembling deviation, movement deviation of the rotary disc and other reasons, the tip position of the pipette generates deviation of three dimensions in different degrees. This offset has the following effect on micropipetting operations:
when liquid is extracted, the position of the tip of the pipette is deviated to cause the pipette to suck air bubbles, and the excessive air bubbles can cause errors in subsequent operations;
when liquid is injected, the position of the tip of the pipette deviates to cause that the liquid cannot be injected to a preset position smoothly, so that the liquid is lost; and
when the turntable rotates, the displacement of the tip of the pipette causes the pipette to collide with the test tube and other parts on the turntable, and the system operation is affected.
It can be seen that pipetting operations and system operation are affected due to the positional offset of the pipette tip.
It is noted that the information disclosed in the background section above is only for enhancement of understanding of the background of the present disclosure, and therefore, may include information that does not constitute prior art that is known to those of ordinary skill in the art.
Disclosure of Invention
In view of the above, the present disclosure discloses a pipetting system and a calibration method thereof to achieve calibration of the pipetting system, and overcome the problem in the prior art that the pipetting operation and the system operation are affected due to the position offset of the pipette tip.
One embodiment of the present disclosure discloses a calibration method of a pipetting system, the calibration method including: capturing at least one first image of a calibration point by an image capturing device; obtaining a first offset of a central point of the image capturing device relative to the calibration point in the first image on a plane, and adjusting the position of the image capturing device according to the first offset to calibrate the central point and the calibration point; capturing at least a second image and a third image of a tip of a pipette of the pipetting system by the calibrated image capturing device, wherein the third image is captured after a focal distance between the tip and the calibrated center point is displaced; obtaining a second offset of the tip in the third image relative to the tip in the second image in the plane; obtaining a focus distance offset of the sharpness value of the third image relative to a preset sharpness value according to the displacement, the variation of the sharpness value of the third image relative to the second image and a preset mapping relation table of the focus distance and the sharpness value; and adjusting the position of the pipette according to the second offset and the focus distance offset, so that the tip and the calibrated central point are calibrated.
In one embodiment of the present disclosure, the step of obtaining a first offset of a center point of the image capturing device with respect to the calibration point in the first image in a plane comprises: obtaining rectangular coordinates of the center point and the calibration point in the first image on the plane parallel to a plane on which a rotating disc of the pipetting system is located; respectively converting the rectangular coordinates of the central point and the rectangular coordinates of the calibration points into polar angles so as to obtain included angles of the polar angles of the central point relative to the polar angles of the calibration points; and obtaining the first offset according to the included angle.
In one embodiment of the present disclosure, the step of obtaining the rectangular coordinates of the calibration point in the first image on the plane comprises: carrying out smooth filtering on the first image to remove noise of the first image; binarizing the first image after smooth filtering to assign values to each pixel point in the first image after smooth filtering; measuring edges of the first binarized image, and marking a plurality of edge points of the first binarized image; carrying out contour calibration on the edge points of the first image after edge measurement to form at least one contour; carrying out roundness detection on each contour, and screening out the contours with roundness values smaller than a preset value; screening the area of the screened outline to screen out an outline of which the area accords with the area of the calibration point; and obtaining the rectangular coordinate of the centroid of the screened outline on the plane as the rectangular coordinate of the calibration point in the first image on the plane.
In one embodiment of the present disclosure, the step of obtaining a second offset of the tip in the third image with respect to the tip in the second image in a plane comprises: respectively obtaining a first rectangular coordinate of the center of the tip in the third image on the plane and a second rectangular coordinate of the center of the tip in the second image on the plane, wherein the plane is perpendicular to the direction of the focal distance; obtaining a pixel offset of the center of the tip in the third image relative to the center of the tip in the second image according to the first rectangular coordinate and the second rectangular coordinate, wherein the rectangular coordinate takes a pixel as a coordinate unit; and converting the pixel offset into a distance offset according to the corresponding relation between a pixel and the distance, wherein the distance offset is used as the second offset.
In an embodiment of the present disclosure, the step of obtaining a first rectilinear coordinate of the center of the tip in the third image on the plane includes: carrying out smooth filtering on the third image to remove noise of the third image; binarizing the third image after smooth filtering to assign values to each pixel point in the third image after smooth filtering; measuring edges of the binarized third image, and marking a plurality of edge points of the binarized third image; carrying out contour calibration on the edge points of the third image after edge measurement to form at least one contour; carrying out roundness detection on each contour, and screening out the contours with roundness values smaller than a preset value; screening the area of the screened outline, and screening out an outline of which the area accords with the area of the tip end; and obtaining a rectangular coordinate of the centroid of the screened contour on the plane as a rectangular coordinate of the center of the tip in the third image on the plane.
In an embodiment of the present disclosure, the step of determining a corresponding relationship between a pixel and a distance includes: capturing an image of an object with a preset size through the image capturing device; and obtaining the corresponding relation between the pixels and the distance according to the number of the pixels of the image of the object and the preset size.
In an embodiment of the disclosure, the step of obtaining a focus distance offset of the sharpness value of the third image relative to a predetermined sharpness value according to the displacement, a variation of the sharpness value of the third image relative to the second image, and a mapping relation table of a predetermined focus distance and sharpness value includes: obtaining the position of the definition value of the third image in the mapping relation table according to the direction of the displacement and the variation of the definition value of the third image relative to the definition value of the second image, wherein the definition values in the mapping relation table are in Gaussian distribution; and according to the position of the definition value of the third image in the mapping relation table, obtaining the focus distance offset of a focus distance corresponding to the definition value of the third image relative to a focus distance corresponding to a preset definition value, wherein the preset definition value is the highest definition value in the mapping relation table.
In an embodiment of the disclosure, the calibration method further includes setting an imaging depth of field of the image capturing device to make a detectable range of the focus distance offset be within a preset distance range, wherein in the capturing the third image, the displacement is greater than the preset distance range.
Another embodiment of the present disclosure discloses a pipetting system, comprising: an image capturing device; and a processor configured to implement, by executing a plurality of executable instructions: capturing at least one first image of a calibration point through the image capturing device; obtaining a first offset of a central point of the image capturing device relative to the calibration point in the first image on a plane, and adjusting the position of the image capturing device according to the first offset to calibrate the central point and the calibration point; capturing at least a second image and a third image of a tip of a pipette of the pipetting system by the calibrated image capturing device, wherein the third image is captured after a focal distance between the tip and the calibrated center point is displaced; obtaining a second offset of the tip in the third image relative to the tip in the second image in the plane; obtaining a focus distance offset of the sharpness value of the third image relative to a preset sharpness value according to the displacement, the variation of the sharpness value of the third image relative to the second image and a preset mapping relation table of the focus distance and the sharpness value; and adjusting the position of the pipette according to the second offset and the focus distance offset, so that the tip and the calibrated central point are calibrated.
In one embodiment of the present disclosure, the pipetting system further comprises: a rotary disc with the image capturing device.
Compared with the prior art, the beneficial effects of this disclosure include at least:
capturing the image of the calibration point by the image capturing device, obtaining a first offset of a central point of the image capturing device relative to the calibration point in the plane dimension, and realizing the calibration of the image capturing device in the plane dimension, so that the calibrated image capturing device is used for positioning in the subsequent calibration step; acquiring images of the tip of the pipette at least twice by the calibrated image acquisition device to obtain a second offset of the tip of the pipette in the plane dimension and a focus distance offset in the focus distance dimension, so as to realize calibration of the tip of the pipette in the plane dimension and the focus distance dimension; thus, the present disclosure utilizes an image capture device to achieve the calibration of the pipetting system, thereby achieving the control of the subsequent pipetting operations and system operation.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and together with the description, serve to explain the principles of the disclosure. It is to be understood that the drawings in the following description are merely exemplary of the disclosure, and that other drawings may be derived from those drawings by one of ordinary skill in the art without the exercise of inventive faculty.
FIG. 1 shows a schematic diagram of a pipetting system in one embodiment of the disclosure;
FIG. 2 is a schematic structural diagram of a pipetting device in one embodiment of the present disclosure;
FIG. 3 is a schematic flow chart illustrating a method of calibrating a pipetting system in one embodiment of the present disclosure;
FIG. 4 is a schematic diagram illustrating an image capturing device capturing a first image of a calibration point according to an embodiment of the present disclosure;
FIG. 5 is a flow chart illustrating obtaining a first offset according to an embodiment of the present disclosure;
FIG. 6 is a schematic flow chart illustrating a process for obtaining rectangular coordinates of calibration points in an embodiment of the present disclosure;
FIG. 7 is a schematic diagram illustrating an embodiment of the present disclosure in which the image capturing device captures a second image of the tip of a pipette;
FIG. 8 is a schematic diagram illustrating an embodiment of the present disclosure in which the image capturing device captures a third image of the tip of the pipette;
FIG. 9 is a flow chart illustrating obtaining a second offset according to an embodiment of the present disclosure;
FIG. 10 is a flowchart illustrating a process of obtaining a first rectilinear coordinate of the center of the tip in the third image according to an embodiment of the present disclosure;
FIG. 11 is a flow chart illustrating a process of obtaining a pixel-to-distance correspondence according to an embodiment of the disclosure;
FIG. 12 is a flow chart illustrating obtaining a focus distance offset in an embodiment of the present disclosure; and
FIG. 13 shows a block schematic diagram of a pipetting system in an embodiment of the disclosure.
Detailed Description
Example embodiments will now be described more fully with reference to the accompanying drawings. Example embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art. The same reference numerals in the drawings denote the same or similar structures, and thus their repetitive description will be omitted.
Fig. 1 illustrates the main structure of a pipetting system in one embodiment, and in some embodiments, the disclosed method of calibrating a pipetting system can be used to calibrate a pipetting system as shown in fig. 1. Referring to fig. 1, the pipetting system includes a carousel 10 and a pipetting device 30. The pipetting device 30 is, for example, a vertical syringe pump, and the pipetting operation is carried out by means of a pipette 31. The turntable 10 is provided with a pipette replacement area 101 and a pipetting area 102.
Fig. 2 shows the main structure of the pipetting device 30 in the pipetting system, the pipetting device 30 is connected with a slide rail 300, and the pipetting device 30 drives the pipette 31 to move along the slide rail 300, so as to realize the pipetting operation. Further, the pipetting system of the embodiment further includes an image capturing device 21 mounted on the turntable 10, and a fixed calibration point 11. The image capturing device 21 and the calibration point 11 are used to calibrate the turntable 10 and the pipette 31 of the pipetting system.
FIG. 3 shows the steps of a method for calibrating a pipetting system in one embodiment. Referring to fig. 3, the calibration method of the pipetting system in this embodiment includes: step S10, capturing at least a first image of a calibration point by an image capturing device; step S20, obtaining a first offset of a center point of the image capturing device relative to the calibration point in the first image, and adjusting the position of the image capturing device according to the first offset to calibrate the center point and the calibration point; step S30, capturing at least a second image and a third image of a tip of a pipette of the pipetting system by the calibrated image capturing device, wherein the third image is captured after a focal distance between the tip and the calibrated center point is shifted; step S40, obtaining a second offset of the tip in the third image relative to the tip in the second image in the plane; step S50, obtaining a focus distance offset of the sharpness value of the third image relative to a predetermined sharpness value according to the displacement, the variation of the sharpness value of the third image relative to the second image, and a predetermined mapping relation table between focus distance and sharpness value; and step S60, adjusting the position of the pipette according to the second offset and the focus distance offset, so that the tip is calibrated with the calibrated center point.
In the embodiment, the image of the calibration point is captured by the image capturing device, the first offset of the central point of the image capturing device relative to the calibration point in the plane dimension is obtained, the calibration of the image capturing device in the plane dimension is realized, and the calibrated image capturing device is used for positioning in the subsequent calibration step; acquiring images of the tip of the pipette at least twice by the calibrated image acquisition device to obtain a second offset of the tip of the pipette in the plane dimension and a focus distance offset in the focus distance dimension, so as to realize calibration of the tip of the pipette in the plane dimension and the focus distance dimension; therefore, the present embodiment utilizes an image capturing device to achieve the calibration of the pipetting system, thereby achieving the control of the subsequent pipetting operation and the system operation.
In the present embodiment, the image capturing device is a microscope camera, and in other embodiments, the image capturing device is a micro camera as long as it has an image capturing function. The calibration point is a fixed reference point, and in this embodiment the calibration point is of the pipetting system, and in other embodiments the calibration point is not of the pipetting system.
Fig. 4 is a schematic diagram illustrating an embodiment of an image capturing device capturing a first image of a calibration point. Referring to fig. 4, in the embodiment, the calibration point 11 is located above the image capturing device 21, and before capturing the image of the calibration point 11 in step S10, the image capturing device 21 is moved to the calibration point 11 and enters the capturing area of the image capturing device 21. In other embodiments, the relative position between the calibration point and the image capturing device is adjusted as long as the condition that the calibration point can enter the capturing area of the image capturing device and the image capturing device performs calibration with reference to the calibration point is satisfied.
FIG. 5 illustrates the main steps for obtaining a first offset in one embodiment. Referring to fig. 5, the step of obtaining a first offset of a center point of the image capturing device with respect to the calibration point in the first image in a plane in step S20 includes: s202, respectively obtaining rectangular coordinates of the central point and the calibration point in the first image on the plane, wherein the plane is parallel to a plane where a rotating disc of the pipetting system is located; s204, respectively converting the rectangular coordinate of the central point and the rectangular coordinate of the calibration point into polar angles to obtain an included angle of the polar angle of the central point relative to the polar angle of the calibration point; and S206, obtaining the first offset according to the included angle.
Referring to fig. 4 and 5, the plane is indicated as X-Y plane, which is parallel to the plane of the image capturing device 21. The plane on which the image capturing device 21 is located is an operation platform for storing and receiving liquid in the pipetting system, for example, in the pipetting system equipped with a turntable, the plane on which the image capturing device 21 is located is the turntable of the pipetting system. In step S202, an X-Y rectangular coordinate system is first established according to the X-Y plane, and then rectangular coordinates of the center point 211 and the calibration point 11 in the first image in the X-Y rectangular coordinate system are respectively obtained. Since the position of the center point 211 is known after the image capturing device 21 is installed, in some embodiments, an X-Y rectangular coordinate system is established with the center point 211 as the origin, and rectangular coordinates of the calibration point 11 in the first image are obtained by projecting the first image to the X-Y rectangular coordinate system, so as to facilitate the calculation of the first offset. In some embodiments, an image of the center point 211 is pre-stored, and the rectangular coordinates of the center point 211 and the rectangular coordinates of the calibration point 11 are obtained by projecting the image of the center point 211 and the first image onto an X-Y rectangular coordinate system, respectively.
The rectangular coordinates of the calibration point 11 are obtained by a center circle contour searching algorithm. Fig. 6 shows the main steps of obtaining the rectangular coordinates of the calibration point in one embodiment, and referring to fig. 6, the step of obtaining the rectangular coordinates of the calibration point in the first image on the plane in step S202 includes: s2021, performing smooth filtering on the first image to remove noise of the first image; s2022, carrying out binarization on the first image after smooth filtering, and assigning a value to each pixel point in the first image after smooth filtering; s2023, performing edge detection on the binarized first image, and marking a plurality of edge points of the binarized first image; s2024, carrying out contour calibration on the plurality of edge points of the first image after edge measurement to form at least one contour; s2025, carrying out roundness detection on each contour, and screening out the contours with the roundness values smaller than a preset value; s2026, area screening is carried out on the screened outline, and an outline with the area meeting the area of the calibration point is screened out; and S2027, obtaining the rectangular coordinate of the centroid of the screened outline on the plane as the rectangular coordinate of the calibration point in the first image on the plane.
Specifically, the binarization in step S2022 is to set the pixels in the smoothed and filtered first image that exceed a preset threshold to be 1, and otherwise to be 0. In this embodiment, the preset threshold is set according to the requirement, and is not limited herein. In the step S2023, the edge detection is performed by using a Canny edge detection algorithm to mark edge points of the binarized first image. The Canny edge detection algorithm is a multi-level edge detection algorithm used to identify actual edges in an image. In the contour calibration of step S2024, the connected edge points detected in the previous step are connected together to form a contour, and since there may be a plurality of contours in the first image, a plurality of groups of connected edge points are detected during edge measurement, and a plurality of contours are indicated during the contour calibration. The roundness detection in step S2025 is performed by the formula "T ═ 4 pi × S/C2"calculate the circularity values of the respective contours indicated at the last step and screen out contours with low circularity values, leaving contours close to a circle. The preset value for evaluating the circularity value is set according to the requirement, and is not limited herein. In the formula "T ═ 4 π S/C2"where T is the roundness value of a contour, S is the area of the contour, and C is the perimeter of the contour. The area S and perimeter C of a profile are obtained by software measurements. SchoolThe area of the collimation point is known, the area screening of step S2026 is performed by calculating the area of each contour left in the last step and screening out a contour whose contour area is closest to the area of the calibration point, and then the rectangular coordinates of the centroid of the contour on the X-Y plane are obtained as the rectangular coordinates of the calibration point 11 by step S2027. Wherein the centroid of a contour is the geometric center of the contour.
Similarly, when the rectangular coordinates of the center point 211 are required to be obtained, a center circle contour searching algorithm is also adopted, and a description thereof is omitted.
After the rectangular coordinates of the center point 211 and the rectangular coordinates of the calibration point 11 are obtained, the rectangular coordinates of the center point 211 and the rectangular coordinates of the calibration point 11 are converted into polar angles, respectively, through step S204. Taking the conversion of the rectangular coordinates of the calibration point 11 into polar angles as an example, the polar angle θ of the calibration point 11 is obtained by the following formula11：
Wherein (x)11，y11) Is the rectangular coordinate of the calibration point 11 in the X-Y rectangular coordinate system, and the X-Y rectangular coordinate system takes one pixel point as a unit coordinate. Similarly, the rectangular coordinate (x) of the center point 211 is used211，y211) To obtain the polar angle theta of the center point 211211And will not be described further herein. Further, the polar angle θ of the center point 211 is set211Minus the polar angle theta of the calibration point 1111Obtaining the polar angle theta of the center point 211211Polar angle theta relative to calibration point 1111As the first offset amount. In the embodiment where the pipetting system is provided with a turntable, as shown in fig. 4, the angle delta theta is one angle of rotation of the turntable in its direction of rotation.
Subsequently, the position of the image capturing device 21 is adjusted according to the first offset, so that the center point 211 and the calibration point 11 are calibrated to compensate the first offset. For example, the turntable of the pipetting system is controlled to rotate by Δ θ such that the center point 211 is aligned with the calibration point 11 in a direction perpendicular to the X-Y plane.
Fig. 7 shows a schematic diagram of the image capturing device capturing a second image of the tip of a pipette in one embodiment, and fig. 8 shows a schematic diagram of the image capturing device capturing a third image of the tip of the pipette in one embodiment. Referring to fig. 7 and 8, in the embodiment, the pipette 31 is located above the image capturing device 21, and before capturing the image of the tip 311 of the pipette 31, the pipette 31 is moved to move the tip 311 of the pipette 31 into the capturing area of the image capturing device 21. In other embodiments, the relative position between the pipette and the image capturing device can be adjusted as long as the condition that the tip of the pipette can enter the capturing area of the image capturing device and the condition that the tip of the pipette is calibrated with reference to the image capturing device is satisfied. The pipette 31 is mounted on a pipetting device 30 of the pipetting system, the pipetting device 30 being, for example, a vertical syringe pump. In fig. 7, when capturing the second image, the center of the tip 311 of the pipette 31 is aligned with the center 211 of the image capturing device 21 in the direction of the Z coordinate axis perpendicular to the X-Y plane, that is, the pipette 31 conforms to the original assembly state, and the center of the tip 311 in the original assembly state is projected to the origin of the X-Y rectangular coordinate system. Or, the center point 211 after calibration is used as the origin of the three-dimensional coordinate system X-Y-Z in fig. 7 and 8, and since the center point 211 is already calibrated with respect to the calibration point 11, the center point 211 is used as the origin of the three-dimensional coordinate system X-Y-Z, which is beneficial to positioning in the subsequent calibration step. In the third image capture of fig. 8, the pipette 31 is re-mounted, the focal distance between the tip 311 and the calibrated center 211 is shifted, and the tip 311 and the calibrated center 211 may be shifted in the X-Y plane.
FIG. 9 shows the main steps for obtaining a second offset in one embodiment. Referring to fig. 9, the step of obtaining a second offset of the tip in the third image relative to the tip in the second image in the plane in step S40 includes: s402, respectively obtaining a first rectangular coordinate of the center of the tip in the third image on the plane and a second rectangular coordinate of the center of the tip in the second image on the plane, wherein the plane is perpendicular to the direction of the focal distance; s404, obtaining a pixel offset of the center of the tip in the third image relative to the center of the tip in the second image according to the first rectangular coordinate and the second rectangular coordinate, wherein the rectangular coordinate uses a pixel as a coordinate unit; and S406, converting the pixel offset into a distance offset according to a corresponding relationship between a pixel and a distance, wherein the distance offset is used as the second offset.
Referring to fig. 7 to 9, the focus distance is in the Z-axis direction, and the second offset is the offset Δ X and Δ Y of the center of the tip 311 in the third image relative to the center of the tip 311 in the second image, i.e., the calibrated center point 211, in the X-Y plane. Because the X-Y rectangular coordinate system takes one pixel point as a unit coordinate, the obtained coordinate offsets delta X and delta Y are pixel offsets. Further, the distance offset corresponding to the pixel offset can be obtained according to the corresponding relationship between the pixel and the distance.
The first rectangular coordinate of the center of the tip in the third image obtained in step S402 is obtained by using a center circle contour search algorithm. Referring to fig. 10, the main steps of obtaining a first rectilinear coordinate of the center of the tip in the third image in one embodiment, and referring to fig. 10, the step of obtaining a first rectilinear coordinate of the center of the tip in the third image in the plane in step S402 includes: s4021, performing smooth filtering on the third image to remove noise of the third image; s4022, binarizing the third image after smooth filtering, and assigning values to each pixel point in the third image after smooth filtering; s4023, measuring edges of the binarized third image, and marking a plurality of edge points of the binarized third image; s4024, carrying out contour calibration on the edge points of the third image after edge measurement to form at least one contour; s4025, performing roundness detection on each contour, and screening out the contours with roundness values smaller than a preset value; s4026, screening the area of the screened outline to obtain an outline with the area conforming to the area of the tip; and S4027, obtaining a rectangular coordinate of the center of the circle of the screened contour on the plane, and taking the rectangular coordinate of the center of the tip in the third image on the plane. The specific principle of each step of the center circle contour searching algorithm is the same as the process of acquiring the rectangular coordinates of the calibration point 11 in the above embodiment, and therefore, the description is not repeated.
As described above, in some embodiments, the second rectangular coordinate of the center of the tip 311 in the second image in the X-Y plane is the origin of the X-Y rectangular coordinate system. In other embodiments, when it is desired to obtain a second rectangular coordinate of the center of the tip 311 in the second image in the X-Y plane, a center circle contour searching algorithm is also used, and will not be further described herein.
In step S406, an object with a known size is photographed by the image capturing device 21 to obtain a corresponding relationship between the pixel and the distance. Fig. 11 shows the main steps of obtaining the corresponding relationship between a pixel and a distance in an embodiment, and referring to fig. 11, the step S406 according to the corresponding relationship between a pixel and a distance includes: s4062, capturing an image of an object with a preset size by the image capturing device; and S4064, obtaining the corresponding relation between the pixel and the distance according to the number of the pixels of the image of the object and the preset size. For example, an image of an object known to be 100 μm long is captured by the image capturing device 21, and the number of pixels of the image of the object is 10, so that a correspondence relationship of 1 pixel to 10 μm is obtained. According to the corresponding relationship between the pixel and the distance, the pixel offset of the tip 311 in the third image relative to the tip 311 in the second image is converted into an offset distance.
For example, in one example, referring to FIGS. 7 and 8, the second rectangular coordinate of the center of the tip 311 in the second image in the X-Y plane is (297, 249) and the first rectangular coordinate of the center of the tip 311 in the third image in the X-Y plane is (240, 257) calculated by the center circle contour search algorithm. The shift Δ X of the tip 311 in the third image on the X-axis is-57 and the shift Δ Y on the Y-axis is 8. Further based on the correspondence between the pixels and the distances, the tip 311 in the third image is shifted by 570 μm in the X-axis and 80 μm in the Y-axis.
The second image and the third image are also used to obtain the focus distance offset of the tip 311. Referring to fig. 12, in an embodiment, the main steps of obtaining the focus distance offset in step S50 include: s502, obtaining the position of the definition value of the third image in the mapping relation table according to the direction of the displacement and the variation of the definition value of the third image relative to the second image, wherein the definition values in the mapping relation table are in Gaussian distribution; and S504, obtaining the focus distance offset of the focus distance corresponding to the sharpness value of the third image relative to a focus distance corresponding to a preset sharpness value according to the position of the sharpness value of the third image in the mapping relation table, wherein the preset sharpness value is the highest sharpness value in the mapping relation table.
Wherein, the mapping relation table of the focus distance and the definition value is preset. Before the liquid transfer system is calibrated, the images of the tips are shot for multiple times through the image capturing device, definition values under different focusing distances are calculated, and a mapping relation table capable of reflecting the corresponding relation between the focusing distances and the definition values is obtained. For example, in one embodiment, the mapping of the focus distance to the sharpness value is shown in Table 1 below:
table 1: the mapping relation table of the focus distance and the definition value is as follows:
wherein the sharpness value is a normalized sharpness value. Because an optimal focusing distance exists between the tip and the central point of the image capturing device, when the distance between the tip and the central point is equal to the optimal focusing distance, the image of the tip taken by the image capturing device has the highest definition value. When the distance between the tip and the center point is increased or decreased relative to the optimal focusing distance, the sharpness value of the image of the tip captured by the image capturing device is decreased relative to the highest sharpness value. That is, the sharpness value of the image exhibits a characteristic of Gaussian distribution (Gaussian distribution) with respect to the focus distance. Under the law, the definition values in the obtained mapping relation table are in a Gaussian distribution. Therefore, when obtaining the focal distance between the tip and the central point, it is necessary to take the image of the tip with the displaced focal distance at least twice to obtain a specific position of the sharpness value in the mapping table. In some embodiments, the pipette is reinstalled multiple times until the correct focal distance between the tip and the center point can be obtained. It should be noted that, in the present embodiment, only a part of the focus distance and the sharpness value are shown in the mapping table, and in other embodiments, the sharpness values on the left and right sides of the highest sharpness value are approximately symmetrically distributed in the mapping table.
Referring to fig. 7 and 8, the displacement is a known quantity in the direction of the Z coordinate axis. By shifting the focus distance between the tip 311 and the center 211, the sharpness values of the two captured images of the tip 311 are changed, so as to obtain a focus distance corresponding to the sharpness value of the third image of the tip 311 as the actual distance between the tip 311 and the center 211. Taking the direction of the Z coordinate axis shown in fig. 7 and 8 as an example, when the tip 311 moves a certain distance along the Z coordinate axis relative to the center 211 of the image capturing device 21, the focus distance between the tip 311 and the calibrated center 211 increases; when the tip 311 moves downward by a certain distance along the Z coordinate axis relative to the center 211 of the image capturing device 21, the focus distance between the tip 311 and the calibrated center 211 decreases.
The variation of the sharpness value of the third image relative to the sharpness value of the second image is a difference of the sharpness value of the third image relative to the sharpness value of the second image. The definition value of the image is obtained by adopting the existing mode. Specifically, there are various algorithms for evaluating the sharpness of an image, and in the spatial domain, the spatial contrast of the image, that is, the gradient difference of the gray features between adjacent pixels, is mainly considered; in the frequency domain, the frequency components of the image are mainly considered, and the high-frequency components of the image which is clearly focused are more, and the low-frequency components of the image which is blurred are more. In one embodiment, the sharpness values of the second image and the third image are obtained by using a Laplace operator, wherein the Laplace operator calculates gradients in the X-axis direction and the Y-axis direction respectively, and the clearer the image is, the higher the gradient value is in the same scene. The Laplace operator has the following formula:
in the formula of the Laplace operator, "src" is a two-dimensional matrix of an original image, and "dst" is a two-dimensional matrix obtained by respectively performing second order skewing and summation on two dimensions "x" and "y" through "src" to find out the edge value of the image by solving the variation in the direction "x" and the direction "y" to obtain "dst" representing the definition value of the image.
In one embodiment, the preset sharpness value is the highest sharpness value 1 in the mapping table, and the corresponding optimal focus distance is 6280 μm. And (3) calculating according to a Laplace operator to obtain a definition value of a second image of the tip to be 0.865822, increasing the focusing distance, moving the pipette upwards by 20 mu m, shooting again to obtain a third image of the tip, and calculating through the Laplace operator to obtain a definition value of the third image to be 0.890014. According to the feature that the sharpness value increases with the increase of the focus distance and the rule of gaussian distribution, it can be known that, with respect to the optimal focus distance, the focus distance between the tip and the center point when the second image and the third image are shot is smaller than the optimal focus distance, and then it is determined that the sharpness value 0.890014 of the third image is located on the left side of the highest sharpness value 1 in the mapping relation table, and the focus distance corresponding to the sharpness value 0.890014 of the obtained third image is 6260 μm. Of course, when moving the pipette, the displacement is also controlled to avoid influencing the subsequent steps due to too large or too small displacement. In some embodiments, the pipette is reinstalled multiple times to obtain a more accurate focus distance between the tip and the center point. Then, a focus distance shift of 6260 μm from 6280 μm corresponding to the preset sharpness value 1 is obtained as a focus distance 0.890014 of the third image, which is 20 μm, as shown by Δ z in fig. 8.
Further, in one embodiment, the method for calibrating a pipetting system further comprises setting the imaging depth of field of the image capturing device such that the detectable range of the focus offset is within a predetermined distance range, wherein the step of capturing the third image is performed with the displacement greater than the predetermined distance range. The imaging depth d of the image capturing device is set by the following formulatot：
In this embodiment, an objective lens with NA of 0.3 and M of 2.66 is used, where NA is the numerical aperture of the objective lens of the image capturing device, M is the magnification of the objective lens, and NA and M are both known quantities. λ is the wavelength of light, and is usually 0.55 μm. n is the refractive index of the medium between the pipette and the objective lens, air in this example, and the refractive index n is 1. e is the minimum distance resolvable by the image capturing device, which is a known quantity, and in this embodiment, e is 2.2 μm. Therefore, the present embodiment calculates the depth d of the image captured by the image capturing devicetot8.86 μm. When the imaging depth of field is 8.86 μm, the focus distance offset larger than 8.86 μm is detectable, i.e. the detectable range of the focus distance offset is larger than 8.86 μm in this embodiment. Therefore, in the step of capturing the third image, the pipette is re-installed so that the focal distance between the tip of the pipette and the center point of the image capturing device is displaced by more than 8.86 μm, thereby detecting the variation of the sharpness value between the second image and the third image of the tip captured by the image capturing device.
Further, after the second offset and the focus distance offset are obtained, the position of the pipette is adjusted to compensate the second offset and the focus distance offset, so that the tip of the pipette is aligned with the calibrated central point in the three-dimensional directions of the X coordinate axis, the Y coordinate axis and the Z coordinate axis, and the calibration of the pipette is completed.
In summary, in the calibration method of the pipetting system in the above embodiment, the image capturing device captures an image of the calibration point to obtain a first offset of the center point of the image capturing device relative to the calibration point on the X-Y plane, so as to calibrate the turntable of the pipetting system where the image capturing device is located on the X-Y plane, and enable the calibrated image capturing device to be used for positioning in the subsequent calibration step; acquiring images of the tip of the pipette at least twice by the calibrated image acquisition device to obtain a second offset of the tip of the pipette on an X-Y plane and a focus distance offset of the tip of the pipette on a Z coordinate axis, so as to realize calibration of the tip of the pipette on the X-Y plane and the Z coordinate axis; therefore, the calibration method of the pipetting system of the present disclosure utilizes an image capturing device to realize the calibration of the rotation direction of the turntable of the pipetting system and the calibration of the three-dimensional direction of the pipette, so as to realize the control of the subsequent pipetting operation and the system operation.
The present disclosure also discloses a pipetting system. Fig. 13 shows the main modules of a pipetting system in an embodiment, and referring to fig. 13, the pipetting system 1 in this embodiment comprises: an image capturing device 21; and a processor 41, the processor 41 configured to implement by executing a plurality of executable instructions: capturing at least one first image of a calibration point by the image capturing device 21; obtaining a first offset of a center point of the image capturing device 21 relative to the calibration point in the first image in a plane, and adjusting the position of the image capturing device 21 according to the first offset to calibrate the center point and the calibration point; capturing at least a second image and a third image of the tip of a pipette 31 of the pipetting system 1 by the calibrated image capturing device 21, wherein the third image is captured after the focal distance between the tip and the calibrated center point is shifted; obtaining a second offset of the tip in the third image relative to the tip in the second image in the plane; obtaining a focus distance offset of the sharpness value of the third image relative to a preset sharpness value according to the displacement, the variation of the sharpness value of the third image relative to the second image and a preset mapping relation table of the focus distance and the sharpness value; and adjusting the position of the pipette 31 according to the second offset and the focus distance offset, so that the tip is calibrated with the calibrated center point. Wherein in some embodiments executable instructions are stored in the processor 41 and in some embodiments in a memory of the pipetting system 1. The specific implementation and principle of the executable instructions refer to the above description of the calibration method embodiment of the pipetting system, and will not be described herein.
In one embodiment, processor 41 is, for example, a Central Processing Unit (CPU). The processor 41 is connected to the image capturing device 21 and the pipette 31 of the pipetting system 1, respectively, to calculate the first offset, the second offset, and the focus distance offset, and control the image capturing device 21 and the pipette 31 to adjust the positions according to the first offset, the second offset, and the focus distance offset, respectively, to calibrate the pipetting system 1.
In summary, the pipetting system of the above embodiment captures the image of the calibration point through the image capturing device, obtains the first offset of the center point of the image capturing device relative to the plane dimension of the calibration point, and realizes the calibration of the image capturing device in the plane dimension, so that the calibrated image capturing device is used for positioning in the subsequent calibration step; acquiring images of the tip of the pipette at least twice by the calibrated image acquisition device to obtain a second offset of the tip of the pipette in the plane dimension and a focus distance offset in the focus distance dimension, so as to realize calibration of the tip of the pipette in the plane dimension and the focus distance dimension; thus, the present disclosure utilizes an image capture device to achieve the calibration of the pipetting system to achieve the control of the subsequent pipetting operations and system operation.
The foregoing is a more detailed description of the present disclosure in connection with specific preferred embodiments, and it is not intended that the specific embodiments of the present disclosure be limited to these descriptions. For those skilled in the art to which the disclosure pertains, several simple deductions or substitutions may be made without departing from the concept of the disclosure, which should be considered as falling within the protection scope of the disclosure.
Claims (8)
1. A method of calibrating a pipetting system, the method comprising:
capturing at least one first image of a calibration point by an image capturing device;
obtaining a first offset of a central point of the image capturing device relative to the calibration point in the first image on a plane, and adjusting the position of the image capturing device according to the first offset to calibrate the central point and the calibration point;
capturing at least a second image and a third image of a tip of a pipette of the pipetting system by the calibrated image capturing device, wherein the third image is captured after a focal distance between the tip and the calibrated center point is displaced;
obtaining a second offset of the tip in the third image relative to the tip in the second image in the plane;
obtaining a focus distance offset of the sharpness value of the third image relative to a preset sharpness value according to the displacement, the variation of the sharpness value of the third image relative to the second image and a preset mapping relation table of the focus distance and the sharpness value; and
and adjusting the position of the pipette according to the second offset and the focus distance offset, so that the tip and the calibrated central point are calibrated.
2. The calibration method of claim 1, wherein the step of obtaining a first offset of a center point of the image capture device from the calibration point in the first image in a plane comprises:
obtaining rectangular coordinates of the center point and the calibration point in the first image on the plane parallel to a plane on which a rotating disc of the pipetting system is located;
respectively converting the rectangular coordinates of the central point and the rectangular coordinates of the calibration points into polar angles so as to obtain included angles of the polar angles of the central point relative to the polar angles of the calibration points; and
and obtaining the first offset according to the included angle.
3. The calibration method of claim 2, wherein the step of obtaining the rectangular coordinates of the calibration point in the first image on the plane comprises:
carrying out smooth filtering on the first image to remove noise of the first image;
binarizing the first image after smooth filtering to assign values to each pixel point in the first image after smooth filtering;
measuring edges of the first binarized image, and marking a plurality of edge points of the first binarized image;
carrying out contour calibration on the edge points of the first image after edge measurement to form at least one contour;
carrying out roundness detection on each contour, and screening out the contours with roundness values smaller than a preset value;
screening the area of the screened outline to screen out an outline of which the area accords with the area of the calibration point; and
and obtaining the rectangular coordinate of the centroid of the screened outline on the plane as the rectangular coordinate of the calibration point in the first image on the plane.
4. The method of claim 1, wherein the step of obtaining a second offset of the tip in the third image relative to the tip in the second image from a plane comprises:
respectively obtaining a first rectangular coordinate of the center of the tip in the third image on the plane and a second rectangular coordinate of the center of the tip in the second image on the plane, wherein the plane is perpendicular to the direction of the focal distance;
obtaining a pixel offset of the center of the tip in the third image relative to the center of the tip in the second image according to the first rectangular coordinate and the second rectangular coordinate, wherein the rectangular coordinate takes a pixel as a coordinate unit; and
and converting the pixel offset into a distance offset according to the corresponding relation between a pixel and the distance, wherein the distance offset is used as the second offset.
5. The calibration method of claim 4, wherein the step of obtaining a first rectilinear coordinate of the center of the tip in the third image with respect to the plane comprises:
carrying out smooth filtering on the third image to remove noise of the third image;
binarizing the third image after smooth filtering to assign values to each pixel point in the third image after smooth filtering;
measuring edges of the binarized third image, and marking a plurality of edge points of the binarized third image;
carrying out contour calibration on the edge points of the third image after edge measurement to form at least one contour;
carrying out roundness detection on each contour, and screening out the contours with roundness values smaller than a preset value;
screening the area of the screened outline, and screening out an outline of which the area accords with the area of the tip end; and
and obtaining the rectangular coordinate of the centroid of the screened contour on the plane as the rectangular coordinate of the center of the tip on the plane in the third image.
6. The calibration method according to claim 4, wherein the step of determining a pixel-to-distance relationship comprises:
capturing an image of an object with a preset size through the image capturing device; and
and obtaining the corresponding relation between the pixels and the distance according to the number of the pixels of the image of the object and the preset size.
7. The calibration method of claim 1, wherein the step of obtaining a focus distance offset of the sharpness value of the third image relative to a predetermined sharpness value according to the displacement, the variation of the sharpness value of the third image relative to the second image, and a predetermined mapping relation between focus distance and sharpness value comprises:
obtaining the position of the definition value of the third image in the mapping relation table according to the direction of the displacement and the variation of the definition value of the third image relative to the definition value of the second image, wherein the definition values in the mapping relation table are in Gaussian distribution; and
and according to the position of the definition value of the third image in the mapping relation table, obtaining the focus distance offset of a focus distance corresponding to the definition value of the third image relative to a focus distance corresponding to a preset definition value, wherein the preset definition value is the highest definition value in the mapping relation table.
8. The calibration method of claim 1, further comprising setting a depth of field of the image capture device such that a detectable range of the focus offset is within a predetermined range, wherein the step of capturing the third image is performed such that the displacement is greater than the predetermined range.
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