CN115774262A - Cover glass thickness detection device, cover glass thickness detection method, electronic device and storage medium - Google Patents
Cover glass thickness detection device, cover glass thickness detection method, electronic device and storage medium Download PDFInfo
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- CN115774262A CN115774262A CN202310093657.6A CN202310093657A CN115774262A CN 115774262 A CN115774262 A CN 115774262A CN 202310093657 A CN202310093657 A CN 202310093657A CN 115774262 A CN115774262 A CN 115774262A
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Abstract
The application discloses cover glass thickness detection equipment, a method, electronic equipment and a storage medium, and relates to the field of optical detection, wherein an emission module emits emission light, the emission light is collimated and expanded to form expanded beam light which is received by a detection module, an objective lens in the detection module forms the expanded beam light into incident light which is focused on a target surface of a cover glass, the incident light is reflected by the target surface to form reflected light, the reflected light is collected by the detection module and transmitted to a detection module to form a converged light spot, the objective lens defocuses the target surface to obtain defocusing amount, the light spot in defocusing is collected by a detector, a processing unit calculates pixel offset of the light spot, a preset offset relation of the target surface is obtained by fitting the pixel offset and the defocusing amount, therefore, the measurement height of the target surface is calculated, thickness detection of the cover glass on a packaged sample chip is achieved, the light spot pixel offset of the target surface is obtained by scanning the whole cover glass, the defocusing amount and the thickness can be calculated by utilizing the preset offset relation, and the detection process is simple.
Description
Technical Field
The present disclosure relates to the field of optical detection technologies, and in particular, to a cover glass thickness detection device, a cover glass thickness detection method, an electronic device, and a storage medium.
Background
In the gene sequencing process, a microscope objective used by a gene sequencing system generally has high resolution, and the microscope objective generally corrects aberration for a cover glass with fixed thickness in the design stage, if the actual thickness of the cover glass is larger than the ideal cover glass thickness when the objective is designed, additional aberration is introduced into the optical system, so that the image quality is deteriorated, and if the actual thickness of the cover glass is smaller than the ideal cover glass thickness when the objective is designed, the aberration of the optical system is over-corrected, so that the image quality is also deteriorated. Therefore, when the incoming material inspection is performed on a biological sample chip, it is necessary to ensure that the thickness variation of the whole sample cover glass meets the requirement of clear imaging, the thickness of the cover glass commonly used in the market is usually dozens of micrometers to hundreds of micrometers, and the thickness variation of the cover glass meeting the requirement is usually required to be ensured to be between several micrometers and dozens of micrometers.
The thickness of the cover glass in the related art is mainly detected by aiming at the thickness of a single piece of glass of the cover glass, so that the detection of the thickness fluctuation in a small range needs to be completed by using a high-precision detection device, the measurement is difficult and the cost is high, and the related detection device is not suitable for the cover glass packaged on a sample chip.
Disclosure of Invention
The present application is directed to solving at least one of the problems in the prior art. To this end, embodiments of the present application provide a cover glass thickness detection apparatus, a cover glass thickness detection method, an electronic apparatus, and a storage medium, which can simply perform thickness detection on a cover glass packaged on a sample chip.
In a first aspect, an embodiment of the present application provides a cover glass thickness detection apparatus, including:
the emitting module comprises a light source, and is used for emitting light and processing the emitting light into collimated light and expanded light;
a detection module comprising: an objective lens, a semi-transmitting and semi-reflecting mirror and a sample chip;
the objective lens is arranged on an optical axis of the beam expanding light and is used for focusing the beam expanding light to form incident light to the upper surface or the lower surface of the cover glass;
the semi-transmitting and semi-reflecting mirror is arranged above the objective lens and is used for transmitting the beam expanding light to the objective lens;
the sample chip is arranged below the objective lens and comprises the cover glass, and the sample chip is used for receiving the incident light of the objective lens and reflecting the incident light to form reflected light;
the detection module comprises a converging mirror and a detector;
the converging mirror is arranged on the optical axis of the reflected light and is used for converging the reflected light to form converging light;
the detector is arranged at a focus behind the converging mirror along the optical axis of the converging light and is used for collecting light spots formed by the converging light;
and the processing unit is used for obtaining the thickness information of the cover glass according to the pixel offset of the light spot and a preset offset relation.
In some embodiments of the present application, the transmitting module further comprises:
the collimating mirror is arranged on the optical axis of the emitted light and is used for collimating the emitted light to obtain collimated light;
the beam expanding lens is arranged on the optical axis of the collimated light and is used for expanding the beam of the collimated light to obtain expanded light;
and the blocking piece is arranged on the optical axis of the beam expanding light and is used for blocking the beam expanding light.
In some embodiments of the present application, the blocking sheet is configured to block the expanded light according to a preset blocking rate.
In some embodiments of the present application, the apparatus further comprises: and the displacement table is used for loading and moving the sample chip according to a preset stepping amount so as to detect the thickness of the cover glass.
In a second aspect, embodiments of the present application further provide a cover slip thickness detection method applied to a cover slip thickness detection apparatus as provided in an embodiment of the first aspect of the present application, where the cover slip includes an upper surface and a lower surface, the method including:
moving the sample chip to a first preset position, acquiring the absolute height of the objective lens when the objective lens and the target surface of the cover glass are focused, and acquiring a first pixel position of the light spot on the detector;
moving the sample chip from the first preset position to a second preset position according to a preset stepping amount to obtain a second pixel position of the light spot on the detector;
selecting a preset offset relationship according to the target surface, wherein the preset offset relationship comprises a first preset offset relationship and a second preset offset relationship;
when the target surface is an upper surface, calculating the defocusing amount of the upper surface of the second preset position according to the first pixel position, the second pixel position and a first preset offset relation, and calculating the measured height of the upper surface according to the defocusing amount of the upper surface and the first absolute height of the objective lens;
when the target surface is a lower surface, calculating the lower surface defocusing amount of a second preset position according to the first pixel position, the second pixel position and a second preset offset relation, and calculating to obtain a lower surface measurement height according to the lower surface defocusing amount and a second absolute height of the objective lens;
and obtaining the thickness of the second preset position of the cover glass according to the upper surface measuring height, the lower surface measuring height and the refractive index.
In some embodiments of the application, before the selecting the preset offset relationship according to the target surface, the method further includes:
controlling the objective lens to be focused on the target surface of the cover glass at a preset initial position, and acquiring a first reference pixel position of the light spot on the detector;
controlling the objective lens and the target surface to defocus, recording reference defocus amounts corresponding to N defocus heights, and acquiring a second reference pixel position of the light spot on the detector at each defocus height, wherein N is an integer greater than 1;
calculating a reference pixel offset corresponding to each reference defocus amount, wherein the reference pixel offset is calculated according to the first reference pixel position and the second reference pixel position corresponding to the reference defocus amount;
when the target surface is an upper surface, fitting according to the reference pixel offset and the reference defocus amount to obtain the first preset offset relation;
and when the target surface is a lower surface, fitting according to the reference pixel offset and the reference defocus amount to obtain the second preset offset relationship.
In some embodiments of the present application, the calculating a reference pixel offset corresponding to each reference defocus amount, where the reference pixel offset is calculated according to the first reference pixel position and the second reference pixel position corresponding to the reference defocus amount, further includes:
squaring the difference between the abscissa of the first reference pixel position and the abscissa of the second reference pixel position to obtain a first result;
squaring the difference value of the vertical coordinate of the first reference pixel position and the vertical coordinate of the second reference pixel position to obtain a second result;
adding the first result and the second result and then squaring to obtain a third result;
and multiplying the third result and the pixel size of the detector to obtain the reference pixel offset corresponding to the reference defocus amount.
In some embodiments of the present application, the obtaining the thickness of the second preset position of the cover glass according to the upper surface measurement height, the lower surface measurement height and the refractive index further comprises:
acquiring the refractive index of the cover glass;
calculating a measurement height difference according to the upper surface measurement height and the lower surface measurement height;
and obtaining the thickness of the second preset position of the cover glass according to the measured height difference and the refractive index.
In a third aspect, embodiments of the present application further provide an electronic device, including a memory and a processor, where the memory stores a computer program, and the processor implements the cover glass thickness detection method according to the embodiments of the second aspect of the present application when executing the computer program.
In a fourth aspect, the present application further provides a computer-readable storage medium, which stores a program, the program being executed by a processor to implement the cover glass thickness detection method according to the second aspect of the present application.
The embodiment of the application at least comprises the following beneficial effects: the embodiment of the application provides cover glass thickness detection equipment, a cover glass thickness detection method, electronic equipment and a storage medium, wherein an emission module in the cover glass thickness detection equipment comprises a light source, emitted light is processed into beam expanding light to be received by a detection module after being emitted, an objective lens in the detection module forms incident light through the beam expanding light transmitted by a half-mirror and focuses the incident light on the upper surface or the lower surface of a cover glass in a sample chip, the upper surface or the lower surface of the cover glass reflects the incident light to form reflected light, the reflected light is received and detected by the detection module through the objective lens and the half-mirror again, the reflected light is collected by a detector to form a light spot after being converged by a converging mirror in the detection module, then a processing unit calculates thickness information of the cover glass according to the pixel offset and a preset offset relation of the light spot, the thickness information of the cover glass of the packaged sample chip is detected in the process only by scanning the cover glass on the whole sample chip, and then the thickness information of the cover glass can be calculated according to the preset offset relation according to the pixel offset of the corresponding position of the upper surface or the lower surface of the cover glass, and the equipment structure and the detection process are simple.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
The above and/or additional aspects and advantages of the present application will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 is a schematic view of a cover slip thickness detection apparatus provided in one embodiment of the present application;
FIG. 2 is a schematic structural diagram of a cover glass thickness detection device provided by an embodiment of the present application;
FIG. 3 is a schematic flow chart diagram of a cover slip thickness detection method provided by an embodiment of the present application;
FIG. 4 is a schematic flow chart of FIG. 3 before step S103;
FIG. 5 is a schematic flowchart of step S203 in FIG. 4;
FIG. 6 is a diagram of a pre-set offset relationship of the top surface of a cover glass provided in an embodiment of the present application;
FIG. 7 is a graph showing the relationship between the height of the upper surface of a cover glass provided in the embodiment of the present application;
FIG. 8 is a graph showing the relationship between the height of the lower surface of a cover glass provided in the embodiment of the present application;
FIG. 9 is a schematic flowchart of step S105 in FIG. 3;
FIG. 10 is a graph showing the thickness measurement of a cover slip according to an embodiment of the present application;
fig. 11 is a schematic structural diagram of an electronic device according to an embodiment of the present application.
Reference numerals: the device comprises an emission module 100, a light source 101, a collimating lens 102, a beam expander 103, a baffle 104, a detection module 200, a half-mirror 201, an objective lens 202, a sample chip 203, a displacement table 204, a detection module 300, a converging lens 301, a detector 302, an electronic device 1000, a processor 1001 and a memory 1002.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative and are only for the purpose of explaining the present application and are not to be construed as limiting the present application.
In the description of the present application, it is to be understood that the positional descriptions, such as the directions of up, down, front, rear, left, right, etc., referred to herein are based on the directions or positional relationships shown in the drawings, and are only for convenience of description and simplification of description, and do not indicate or imply that the referred device or element must have a specific direction, be constructed and operated in a specific direction, and thus, should not be construed as limiting the present application.
In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and larger, smaller, larger, etc. are understood as excluding the present number, and larger, smaller, inner, etc. are understood as including the present number. If there is a description of first and second for the purpose of distinguishing technical features only, this is not to be understood as indicating or implying a relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of technical features indicated.
In the description of the present application, unless otherwise expressly limited, terms such as set, mounted, connected and the like should be construed broadly, and those skilled in the art can reasonably determine the specific meaning of the terms in the present application by combining the detailed contents of the technical solutions.
The biological sample chip used in gene sequencing is provided with a cover glass, a flow channel is arranged between the cover glass and a glass slide, a method of modifying a primer on the upper surface of the flow channel or the lower surface of the flow channel is used for capturing a DNA template chain to be detected, and the gene sequencing is completed by utilizing a technology of synthesizing and sequencing at the same time and combining a micro-optical system. In the gene sequencing process, a microscope objective used by a gene sequencing system generally has high resolution, and the microscope objective generally corrects aberration for a cover glass with fixed thickness in the design stage, if the actual thickness of the cover glass is larger than the ideal cover glass thickness when the objective is designed, additional aberration is introduced into the optical system, so that the image quality is deteriorated, and if the actual thickness of the cover glass is smaller than the ideal cover glass thickness when the objective is designed, the aberration of the optical system is over-corrected, so that the image quality is also deteriorated. Therefore, when the incoming material inspection is performed on a biological sample chip, it is necessary to ensure that the thickness variation of the whole sample cover glass meets the requirement of clear imaging, and the thickness of the cover glass commonly used in the market is usually dozens of micrometers to hundreds of micrometers, so the thickness variation of the cover glass meeting the requirement is usually required to be ensured to be between several micrometers to dozens of micrometers. The related art detection of the thickness of the cover glass mainly aims at the detection of the thickness of a single piece of glass of the cover glass, the detection of the thickness fluctuation in such a small range needs to be completed by using a high-precision detection device, the measurement is difficult and the cost is high, and the related detection device is not suitable for the cover glass packaged on a sample chip.
Based on this, an embodiment of the present application provides a cover slip thickness detection device, a method, an electronic device, and a storage medium, where an emission module in the cover slip thickness detection device includes a light source, the emission module emits emission light and processes the emission light into beam-expanded light to be received by a detection module, an objective lens in the detection module focuses the beam-expanded light transmitted by a half-mirror onto an upper surface or a lower surface of a cover slip in a sample chip, the upper surface or the lower surface of the cover slip reflects the emission light to form reflection light, the reflection light is received and detected by the detection module through the objective lens and the half-mirror again, the collection mirror in the detection module collects the reflection light and then is collected by a detector to form a light spot, then a processing unit calculates thickness information of the cover slip according to a pixel offset and a preset offset relationship of the light spot, in the process, the cover slip of the packaged sample chip is subjected to thickness detection by only scanning the cover slip on the entire sample chip, and then the thickness information of the cover slip is obtained according to the preset offset relationship according to the pixel offset amount of the light spot on the corresponding position of the upper surface or the lower surface of the cover slip, and the detection process is simple.
Fig. 1 is a schematic view of a cover glass thickness detection device provided in an embodiment of the present application. In the embodiment, the light spot pixel offset of different positions of the upper surface and the lower surface of the cover glass in the sample chip is calculated, so that the thickness of the cover glass for measuring the defocusing amount is calculated according to a preset offset relation.
Referring to fig. 1, the cover glass thickness detecting apparatus includes:
the emitting module 100 includes a light source 101, and the emitting module 100 is configured to emit emitting light and process the emitting light into collimated light and expanded light.
In some embodiments, the light source 101 may be a laser, which has the advantages of high brightness, good directivity, good monochromaticity, good coherence, or may be a mercury lamp, a xenon lamp, or an argon lamp, which is not limited in this embodiment. It can be understood that the emitted light from the light source 101 has a certain divergence angle, so that the emission module 100 is further required to further process the emitted light, and the collimated light is collimated to form collimated light, and then the collimated light is expanded to form expanded light before being emitted.
A detection module 200, comprising: an objective lens 202, a half mirror 201 and a sample chip 203. Specifically, the objective lens 202 is disposed on an optical axis of the beam expanding light and is configured to focus the beam expanding light to form incident light on an upper surface or a lower surface of the cover glass, the half-transmitting mirror 201 is disposed above the objective lens 202 along the optical axis of the beam expanding light and is configured to transmit the beam expanding light to the objective lens 202, the sample chip 203 is disposed below the objective lens 202 along the optical axis of the beam expanding light, and the cover glass is configured to receive the incident light formed by the beam expanding light converged by the objective lens 202 and reflect the incident light to form reflected light.
In some embodiments, after the cover glass of the sample chip 203 reflects the incident light, the reflected light continues to be collected by the objective lens 202 and then reflected by the half mirror 201.
A detection module 300, comprising: a converging mirror 301 and a detector 302. Specifically, the converging mirror 301 is disposed on an optical axis of the reflected light for converging the reflected light to form a converging light, and the detector 302 is disposed at a focal point behind the converging mirror 301 along the optical axis of the converging light for collecting a light spot formed by the converging light.
The cover glass thickness detection device of the embodiment of the application further comprises a processing unit (not shown in the figure), and the processing unit is used for obtaining the thickness information of the cover glass according to the pixel offset of the light spot and the preset offset relation. It can be understood that the light spot is formed by collecting and collecting the incident light reflected by the cover glass by the detection module 300, and the cover glass height variation at the corresponding position can be calculated according to the preset offset relationship, so that the thickness information can be calculated, and the measurement process is simple.
Referring to fig. 2, in some embodiments of the present application, the transmitting module 100 further comprises: a collimating lens 102, a beam expanding lens 103 and a baffle plate 104. Specifically, the collimating lens 102 is arranged below the light source 101 along an optical axis of emitted light, and is used for collimating the emitted light emitted by the light source 101 to obtain collimated light, the beam expanding lens 103 is arranged below the collimating lens 102 along the optical axis of the collimated light to expand the beam of the collimated light, and the blocking piece 104 is arranged below the beam expanding lens 103 along the optical axis of the expanded beam of the beam expanding lens to block the expanded beam of the beam expanding light.
It should be understood that the collimating mirror 102 is an instrument for CO2 laser and infrared optical systems, and in particular, the collimating mirror 102 is used in various optical measurement systems, and is often used for collimating an optical path, so that a divergent optical path is changed into a parallel optical path, and by matching different optical elements, the beam collimation of different measurement processes can be optimized. The beam expander 103 can change the diameter of the light beam, and the divergence means that the light wave is spread at a certain angle in the space propagation process, specifically, the beam expander 103 expands the diameter of the light beam and reduces the divergence angle of the light beam.
Specifically, the beam expander 103 may be a galilean beam expander, which generally includes an input concave lens and an output convex lens, where the input lens transmits a virtual focal length light beam to the output lens, or a keplerian beam expander, which generally has one convex lens as an input lens and another convex lens as an output lens, where the input lens transmits a real focal length focused light beam to an output element, or other systems capable of achieving the purpose of beam expansion, and this embodiment is not limited thereto.
It can be understood that the half-transmitting and half-reflecting mirror is an optical element which is formed by plating a half-reflecting film on optical glass and changes the original transmission and reflection proportion of an incident beam, and the film plating layer can increase the reflection, increase the light intensity, increase the reflection and reduce the light intensity. Transflective means that the film has a transmittance and a reflectance of about 50% each, and that when light passes through the film, the transmitted light and the reflected light each account for about 50%.
In some embodiments, the blocking plate 104 is disposed between the beam expander 103 and the half mirror 201 or between the beam expander 103 and the half mirror for blocking a part of the expanded beam according to a preset blocking ratio. Illustratively, when the predetermined shielding rate is 50%, the blocking plate 104 is used to shield half of the expanded beam light, and the remaining half of the expanded beam light can be transmitted to the objective lens 202 through the half mirror 201.
In some embodiments, the cover slip thickness detection apparatus further includes a displacement stage 204 for loading and moving the sample chip 203 by a preset step amount for cover slip thickness detection. Specifically, the displacement stage 204 drives the sample chip 203 to move, so that the detector 302 detects the thickness of the cover glass at different positions. It can be understood that, before the process of performing thickness detection on the cover glass, a preset offset relationship needs to be established, and at this time, the same position on the upper surface or the lower surface of the cover glass needs to be focused and then defocused, so that the method can be realized by finely adjusting the displacement stage 204 up and down, and can also be realized by finely adjusting the objective lens 202 up and down, which is not limited in the present application.
It can be understood that the displacement stage 204 is configured to move the sample chip 203 by a preset step amount, for example, the preset step amount distance is 10 micrometers, and the displacement stage 204 moves the loaded sample chip 203 by 10 micrometers each time, so that after each movement, height detection can be performed on different positions of the target surface of the cover glass, specifically, when the target surface is an upper surface, detection on the upper surface of the whole cover glass is performed by movement of the displacement stage 204, and when the target surface is a lower surface, detection on the lower surface of the whole cover glass is performed, and then calculation of the measurement height is performed according to the height variation of the upper surface and the lower surface, so as to perform thickness measurement on the whole cover glass.
Referring to the schematic structural diagram of the cover slip thickness detection apparatus of fig. 2, in some embodiments of the present application, when acquiring the height variation of the top surface of the cover slip, it is necessary to measure different positions of the top surface of the cover slip. Specifically, the emitted light emitted by the light source 101 passes through the collimator lens 102, the collimator lens 102 collimates the emitted light, so that divergent light is converted into collimated light, the collimated light is processed by the beam expander lens 103 to form expanded light, the stop piece 104 shields the expanded light according to a preset shielding rate of 50%, the remaining 50% of the expanded light is emitted to the objective lens 202 through the half mirror 201 to form incident light, the objective lens 202 focuses the incident light to the upper surface of the cover glass on the sample chip 203, reflected light formed by reflecting the incident light on the upper surface of the cover glass is collected by the objective lens 202 to the half mirror 201, the reflected light is reflected by the half mirror 201 and then passes through the condenser lens 301, the condenser lens 301 collects the reflected light to form converged light to the detector 302, the detector 302 collects light spots formed by focusing, the processing unit calculates corresponding pixel offset according to the positions of the light spots, and then calculates corresponding defocus amount based on a preset offset relationship, so as to obtain height information of the upper surface of the cover glass.
It can be understood that the process of obtaining the height variation at each position of the lower surface of the cover glass is similar to the above process, specifically, the objective lens 202 focuses incident light onto the lower surface of the cover glass on the sample chip 203, reflected light formed by reflecting the incident light on the lower surface of the cover glass is collected by the objective lens 202 and reaches the half mirror 201, the reflected light is reflected by the half mirror 201 and then passes through the converging mirror 301, the converging mirror 301 converges the reflected light to form converging light to the detector 302, the detector 302 collects light spots formed by converging and focusing the light, the processing unit calculates a corresponding pixel offset according to the positions of the light spots, and then calculates a corresponding defocus amount based on a preset offset relationship, thereby obtaining the height information of the lower surface of the cover glass.
In some embodiments, the sample chip 203 is moved by the displacement stage 204 by a preset step amount from the initial position to the end position, and for example, the motor drives the displacement stage 204 to scan the detector from the upper left corner of the sample chip 203 to the lower right corner of the sample chip 203 by the preset step amount in the horizontal direction, so as to obtain the light spot information on the detector when the cover glass is at different positions, and the thickness variation of the cover glass is reversely calculated by analyzing the light spot position variation on the detector. Therefore, the upper surface and the lower surface of the cover glass are scanned and detected respectively, according to the preset offset relation, after the corresponding defocusing amount is calculated through the pixel offset of the corresponding position, the height variation of the upper surface and the lower surface of the cover glass is obtained, and then the corresponding measurement height is obtained through calculation, so that the thickness information of the cover glass is obtained, the thickness detection of the cover glass packaged in the sample chip 203 can be completed, the cost is effectively reduced, and the detection process is simple and accurate.
Embodiments of the present invention further provide a cover slip thickness detection method, which can be applied to the cover slip thickness detection apparatus described above, and as shown in fig. 3, in some embodiments of the present application, the cover slip thickness detection method includes, but is not limited to, the following steps S101 to S105.
Step S101, moving the sample chip to a first preset position, obtaining the absolute height of the objective lens when the objective lens and the target surface of the cover glass are focused, and obtaining a first pixel position of a light spot on the detector.
In some embodiments, the first preset position is an initial position for performing scanning detection on the sample chip 203, and for example, the scanning may be started from the upper left corner of the sample chip 203, that is, the first preset position is the upper left corner of the sample chip 203, when the detector 302 images the upper left corner of the cover glass target surface. It can be understood that the first preset position may also be the upper right corner, the lower left corner, or any position of the sample chip 203, which is not limited in this embodiment of the present application.
In some embodiments, the target surface is the top or bottom surface of the coverslip, specifically, the absolute height of the objective lens 202 when the objective lens 202 is in focus with the coverslip target surface is taken, denoted as D, and the first pixel position of the spot on the detector 302 is taken, denoted as (i) 1 ，j 1 ). Specifically, when the target surface is an upper surface, the absolute height of the objective lens 202 at this time is a first absolute heightIs marked as D 1 When the target surface is the lower surface, the absolute height of the objective lens 202 is a second absolute height, denoted as D 2 . It can be understood that the detector 302 converts the optical signal into an electrical signal, and the processing unit extracts the positions of all the light spot pixel points and the intensities thereof.
And S102, moving the sample chip from the first preset position to a second preset position according to a preset stepping amount, and obtaining a second pixel position of the light spot on the detector.
In some embodiments, during the process of detecting the upper surface or the lower surface of the cover glass, each position of the whole cover glass needs to be scanned, specifically, with the preset step amount of the displacement stage 204 as the unit of each detection, the displacement stage 204 moves once according to the preset step amount, and the cover glass in the sample chip 203 is detected once, so as to obtain the height variation of the cover glass at the corresponding position.
It is understood that the sample chip 203 starts the cover glass thickness detection from the first preset position and moves to the second preset position according to the preset stepping amount, specifically, the second preset position changes with the movement of the displacement stage 204 until the complete Zhang Gai slide is scanned, for example, when the upper left corner of the sample chip 203 is taken as the first preset position, the last second preset position is the lower right corner of the sample chip 203, or the scanning detection starts from the upper right corner of the sample chip 203 and ends at the lower left corner of the sample chip 203, which may also be S-shaped scanning, and the upper left corner of the sample chip 203 is taken as the first preset position, if an odd number row is scanned, the last second preset position is the lower right corner of the sample chip 203, if an even number row is scanned, the last second preset position is the lower left corner of the sample chip 203, which is not limited in this application.
In some embodiments, during each predetermined step of the movement, the pixel position of the light spot imaged by the detector 302 at the second predetermined position on the target surface is different from the first predetermined position, and the second pixel position of the corresponding light spot on the detector 302 is obtained and is marked as (i) 2 ，j 2 ）。
Step S103, selecting a preset offset relationship according to the target surface, wherein the preset offset relationship comprises a first preset offset relationship and a second preset offset relationship.
In some embodiments, the first predetermined offset relationship is selected when the target surface of the cover slip is an upper surface and the second predetermined offset relationship is correspondingly selected when the target surface of the cover slip is a lower surface.
Step S104, when the target surface is an upper surface, calculating the defocusing amount of the upper surface of a second preset position according to the first pixel position, the second pixel position and a first preset offset relation, and calculating the measured height of the upper surface according to the defocusing amount of the upper surface and the first absolute height of the objective lens; and when the target surface is a lower surface, calculating the lower surface defocusing amount of a second preset position according to the first pixel position, the second pixel position and a second preset offset relation, and calculating the lower surface measurement height according to the lower surface defocusing amount and the second absolute height of the objective lens.
In some embodiments, the pixel offset of the same light spot may be calculated according to the corresponding pixel position of the same light spot in the focused state and the defocused state. It can be understood that the first pixel position and the second pixel position correspond to a pixel position with the highest pixel intensity value on the light spot at the detection time, or correspond to a pixel position with the second highest pixel intensity value on the light spot, which is not limited in this embodiment.
In some embodiments, when the target surface is an upper surface, the pixel offset amount is calculated according to the first pixel position and a second pixel position of a second preset position, then the defocus amount of the second preset position of the upper surface can be calculated according to the first preset offset relationship, and finally the upper surface measurement height can be obtained according to the defocus amount and the first absolute height of the objective lens 202. Correspondingly, when the target surface is a lower surface, the pixel offset is calculated according to the first pixel position and the second pixel position of the second preset position, then the defocus amount of the second preset position of the lower surface can be calculated according to the second preset offset relationship, and finally the lower surface measurement height can be obtained according to the defocus amount and the second absolute height of the objective lens 202.
And step S105, obtaining the thickness of the second preset position of the cover glass according to the upper surface measuring height, the lower surface measuring height and the refractive index.
In some embodiments, the thickness of the upper and lower surfaces of the second preset position of the cover glass can be calculated according to the measured height of the upper surface of the second preset position, the measured height of the lower surface of the second preset position and the refractive index of the cover glass, so that the cover glass thickness detection of the second preset position is completed. It can be understood that, the displacement stage 204 drives the sample chip 203 to perform two complete scans, the first complete scan can obtain the measured height of each position on the upper surface of the cover glass, the second complete scan can obtain the measured height of each position on the lower surface, then the thickness of the cover glass at the corresponding position is obtained through calculation, and the calculation process is repeated for each position, so as to obtain the thickness information of the whole cover glass.
Referring to fig. 4, in some embodiments of the present application, a preset offset relationship needs to be obtained by fitting N defocus amounts and pixel offset amounts, and therefore, before the step S103, the following steps S201 to S204 may be further included, but are not limited thereto.
Step S201, controlling the objective lens to focus on the target surface of the cover glass at a preset initial position, and acquiring a first reference pixel position of a light spot on the detector.
In some embodiments, the data used to fit the preset offset relationship may be any position of the cover glass target surface, i.e., a preset initial position. It should be understood that if the target surface is the top surface of the cover slip, the first reference pixel position when the acquisition objective lens 202 is focused on the top surface of the cover slip at the preset initial position is noted as (i) 1 ，j 1 ) Correspondingly, if the target surface is the lower surface of the cover glass, the first reference pixel position (i) at which the objective lens 202 is focused with the lower surface of the cover glass at the preset initial position is acquired 1 ，j 1 ）。
Step S202, controlling the objective lens and the target surface to carry out defocusing, recording reference defocusing amount corresponding to N defocusing heights, and acquiring a second reference pixel position of a light spot on the detector at each defocusing height.
In some embodiments, after controlling the objective lens 202 to be out of focus with respect to the target surface at the preset initial position, reference defocus amounts corresponding to N different defocus heights are recorded as d, and a second reference pixel position of the light spot on the detector 302 is obtained at each defocus height as (i) 2 ，j 2 ). Illustratively, different defocus heights are adjusted for the target surface at the preset initial position, i.e. the upper surface or the lower surface, for example, 5um, 10um, 15um, and so on are defocused respectively. It can be understood that N reference defocus amounts and corresponding N second reference pixels are obtained for fitting a preset offset relationship curve, where N may be an integer greater than 1, such as 5 or 6, and the application does not limit this.
It should be understood that controlling the objective lens 202 out of focus with the target surface may be accomplished by moving either the translation stage 204 or the objective lens 202.
In step S203, a reference pixel shift amount corresponding to each reference defocus amount is calculated.
In some embodiments, the formula is calculated by the distance of two points on the coordinates, passing the first pixel location (i) 1 ，j 1 ) Second pixel position (i) corresponding to each reference defocus amount 2 ，j 2 ) The reference pixel offset corresponding to each reference defocus amount can be calculated, and further, the first pixel position (i) 1 ，j 1 ) And N second pixel positions (i) 2 ，j 2 ) N reference pixel offsets may be calculated.
Step S204, when the target surface is an upper surface, fitting is carried out according to the reference pixel offset and the reference defocusing amount to obtain a first preset offset relation; and when the target surface is the lower surface, fitting according to the reference pixel offset and the reference defocus amount to obtain a second preset offset relation.
In some embodiments, the pixel offset amount changes monotonically with the defocus amount, and a specific fitting curve equation of the preset offset relationship of the target surface can be obtained by fitting the N reference pixel offset amounts and the N reference defocus amounts of the target surface. It is understood that R may be used 2 = regressionAnd evaluating the goodness of fit of the fitting curve by the deviation/total deviation, wherein the goodness of fit is the degree of fit of the regression curve to the observed value. The statistic of metric goodness of fit is the coefficients of decision (also called determination coefficients) R. The closer the value of the maximum value of R1,R is to 1, the better the degree of fitting of the regression curve to the observed value is, the better; conversely, the smaller the value of R, the worse the fitting degree of the regression straight line to the observed value.
Referring to fig. 5, in some embodiments of the present application, the step S203 may further include, but is not limited to, the following steps S301 to S304.
Step S301, a difference between the abscissa of the first reference pixel position and the abscissa of the second reference pixel position is squared to obtain a first result.
In some embodiments, the first reference pixel location coordinate is noted as (i) 1 ，j 1 ) So that its abscissa is i 1 Correspondingly, the second reference pixel coordinate is expressed as (i) 2 ，j 2 ) So that its abscissa is i 2 . To i is to 1 And i 2 Is squared to obtain a first result, i.e., (i) 1 -i 2 ) 2 。
Step S302, a difference between the ordinate of the first reference pixel position and the ordinate of the second reference pixel position is squared to obtain a second result.
In some embodiments, the first reference pixel location coordinate is noted as (i) 1 ，j 1 ) So that its ordinate is j 1 Correspondingly, the second reference pixel coordinate is expressed as (i) 2 ，j 2 ) So that its ordinate is j 2 . To j is paired 1 And j 2 Is squared to obtain a second result, i.e., (j) 1 -j 2 ) 2 。
Step S303, add the first result and the second result and then square the result to obtain a third result.
In some embodiments, for the first result (i) 1 -i 2 ) 2 And a second result (j) 1 -j 2 ) 2 Add and square to obtain a third result, i.e.sqrt((i 1 -i 2 ) 2 +(j 1 -j 2 ) 2 )。
And step S304, multiplying the third result and the pixel size of the detector to obtain a reference pixel offset corresponding to the reference defocus amount.
In some embodiments, the pixel size of detector 302 is denoted as P, for a third result sqrt ((i) 1 -i 2 ) 2 +(j 1 -j 2 ) 2 ) Multiplying the pixel size P of the detector to obtain a reference pixel offset corresponding to the reference defocus amount, namely sqrt ((i) 1 -i 2 ) 2 +(j 1 -j 2 ) 2 ) P, which is denoted S, so S = sqrt ((i) 1 -i 2 ) 2 +(j 1 -j 2 ) 2 )*P。
In some embodiments, the reference pixel shift amount S varies monotonically with the reference defocus amount d, and the preset shift relationship of S-d is obtained by fitting, for example, referring to fig. 6, the preset shift relationship on the upper surface of the cover glass is d =0.00843s +0.1433, and R thereof is =0.00843s +0.1433 2 =0.9997, which shows that the curve has a high degree of fitting, specifically, the general formula relationship of the predetermined offset relationship is y = a + b x, the plot is in a bold manner without weighting, the intercept is 0.1433 ± 0.03664, the slope is 0.00843 ± 4.88122, and the sum of the squares of the residuals is 0.13281.
The displacement stage 204 is used for driving the sample chip 203 to move according to a preset stepping amount, so as to scan the upper surface of the whole cover glass, then the defocusing amount D of the cover glass at different positions in the sample chip 203 is calculated by calculating the pixel offset amount S of light spots at different positions and using a fitted preset offset relation curve, for example, as shown in fig. 6, when the pixel offset amount of the light spots is-600 microns, D =0.00843 (-600) +0.1433= -4.9147 is calculated through a preset offset relation, so that the defocusing amount at the position is-4.9147 um, the defocusing amount D is the height variation amount of the upper surface of the cover glass, and according to a first absolute height D of the objective lens 202 1 The measured height of the current position of the upper surface, i.e. | D, can be calculated 1 +d|=|D 1 4.9147 in the purple sugar um. Thereby passing N in advanceThe preset offset relation of the target surface is obtained by fitting the reference pixel offset and the N reference defocusing amounts, calibration fitting is carried out only when the target surface is used for the first time, the target surface can be directly used in the subsequent cover glass thickness detection process, and the detection method is simple.
Illustratively, this is shown with reference to FIG. 7, which is a graph of the height change of the top surface of the cover glass. Correspondingly, the same fitting process is performed on the lower surface of the cover glass to obtain a second preset offset relationship, as shown in fig. 8, the second preset offset relationship is a defocus amount d calculated by using the pixel offset S at different positions, that is, a height change of the lower surface of the cover glass.
Referring to fig. 9, in some embodiments of the present application, the step S105 may further include, but is not limited to, the following steps S401 to S403.
In step S401, the refractive index of the cover glass is acquired.
In some embodiments, the refractive index of the coverslip is taken and is denoted as n.
Step S402, calculating a measurement height difference according to the upper surface measurement height and the lower surface measurement height.
In some embodiments, the upper surface measurement height is defined by a first absolute height D of the objective lens 202 1 And defocus amount d of upper surface 1 Is calculated as D 1 +d 1 Correspondingly, the lower surface measurement height is determined by the second absolute height D of the objective lens 202 2 And defocus amount d of the lower surface 2 Is calculated as D 1 +d 1 . It will be appreciated that the difference in measured height, i.e. | D, can be calculated by taking the difference between the measured height of the upper surface and the measured height of the lower surface 1 +d 1 -D 1 -d 1 |。
And S403, obtaining the thickness of the second preset position of the cover glass according to the measured height difference and the refractive index.
In some embodiments, the refractive index n and the measured height difference | D 1 +d 1 -D 2 -d 2 Multiplying | to obtain the thickness of the cover glass at the second preset position, i.e. | D 1 +d 1 -D 2 -d 2 N, denoted as THK, i.e. THK = | D 1 +d 1 -D 2 -d 2 | n. Referring to fig. 10, the thickness of the whole cover glass is scanned and detected, the thickness specification of the cover glass is 170 micrometers, if the actual thickness is within 161.5-178.5 micrometers according to the specification of ± 5%, the cover glass can be considered as qualified, as can be seen from the figure, the actual detection thickness interval at each position is 168-176 micrometers, which meets the feeding detection specification, thereby completing the thickness detection of the cover glass.
Fig. 11 shows an electronic device 1000 provided in an embodiment of the present application. The electronic device 1000 includes: a processor 1001, a memory 1002 and a computer program stored on the memory 1002 and executable on the processor 1001 for performing the above-described cover glass thickness detection method when the computer program is run.
The processor 1001 and the memory 1002 may be connected by a bus or other means.
The memory 1002, as a non-transitory computer readable storage medium, may be used to store non-transitory software programs and non-transitory computer executable programs, such as the coverslip thickness detection methods described in the embodiments of the present application. The processor 1001 implements the coverslip thickness detection method described above by running a non-transitory software program and instructions stored in the memory 1002.
The memory 1002 may include a storage program area and a storage data area, wherein the storage program area may store an operating system, an application program required for at least one function; the storage data area may store data for performing the cover slip thickness detection method described above. Further, the memory 1002 may include a high speed random access memory 1002, and may also include a non-transitory memory 1002, such as at least one storage device memory device, flash memory device, or other non-transitory solid state memory device. In some embodiments, the memory 1002 may optionally include memory 1002 located remotely from the processor 1001, and such remote memory 1002 may be coupled to the electronic device 1000 via a network. Examples of such networks include, but are not limited to, the internet, intranets, local area networks, mobile communication networks, and combinations thereof.
Non-transitory software programs and instructions necessary to implement the above-described coverslip thickness detection method are stored in the memory 1002 and, when executed by the one or more processors 1001, perform the above-described coverslip thickness detection method, e.g., performing method steps S101 to S105 in fig. 3, method steps S201 to S204 in fig. 4, method steps S301 to S304 in fig. 5, and method steps S401 to S403 in fig. 6.
The embodiment of the application also provides a storage medium which is a computer readable storage medium and stores a computer program, and the computer program is executed by a processor to realize the cover glass thickness detection method. The memory, which is a non-transitory computer readable storage medium, may be used to store non-transitory software programs as well as non-transitory computer executable programs. Further, the memory may include high speed random access memory, and may also include non-transitory memory, such as at least one disk storage device, flash memory device, or other non-transitory solid state storage device. In some embodiments, the memory optionally includes memory located remotely from the processor, and these remote memories may be connected to the processor through a network. Examples of such networks include, but are not limited to, the internet, intranets, local area networks, mobile communication networks, and combinations thereof.
The cover glass thickness detection device and method, the electronic device and the storage medium provided by the embodiment of the application, an emission module in the cover glass thickness detection device emits emission light and then processes the emission light into collimated light and beam-expanded light to be received by a detection module, an objective lens in the detection module forms the beam-expanded light into incident light and focuses the incident light on a target surface of a cover glass in a sample chip, the target surface of the cover glass reflects the incident light to form reflected light, the reflected light is received and detected by a detection module through the objective lens again, a converging mirror in the detection module converges the reflected light to form converging light and then is collected by a detector to form light spots, a displacement table or the objective lens is moved to enable the target surface of the cover glass to be out of focus, then the light spots in out-of-focus states at corresponding positions are collected through the detector, pixel offset of the light spots is calculated through a processing unit, a preset offset relation of the target surface is obtained through fitting according to the previous pixel offset and out-of focus, and thickness information of each position of the target surface can be calculated. And converting the height information of the upper surface and the lower surface of the cover glass into light spot position information on the detector, and reversely calculating the thickness variation of the cover glass by analyzing the light spot position change on the detector. Thickness detection is carried out to the coverslip of the sample chip that encapsulates at this in-process, and equipment structure is simple, effectively the cost is reduced, only need obtain predetermineeing the skew relation according to a small amount of facula pixel offset and defocus volume fitting when using for the first time, through scanning whole coverslip, the facula pixel offset that obtains the target surface position utilizes predetermineeing the skew relation and can calculate its thickness, and the testing process is simple.
The above-described embodiments of the apparatus are merely illustrative, wherein the units illustrated as separate components may or may not be physically separate, i.e. may be located in one place, or may also be distributed over a plurality of network elements. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of the present embodiment.
One of ordinary skill in the art will appreciate that all or some of the steps, systems, and methods disclosed above may be implemented as software, firmware, hardware, and suitable combinations thereof. Some or all of the physical components may be implemented as software executed by a processor, such as a central processing unit, digital signal processor, or microprocessor, or as hardware, or as an integrated circuit, such as an application specific integrated circuit. Such software may be distributed on computer readable media, which may include computer storage media (or non-transitory media) and communication media (or transitory media). The term computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data, as is well known to those skilled in the art. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital Versatile Disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, storage device storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can accessed by a computer. In addition, communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media as is well known to those skilled in the art.
It should also be appreciated that the various implementations provided in the embodiments of the present application can be combined arbitrarily to achieve different technical effects. While the present application has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (11)
1. A cover glass thickness detection apparatus, comprising:
the emitting module comprises a light source, and is used for emitting light and processing the emitting light into collimated light and expanded light;
a detection module comprising: an objective lens, a semi-transparent and semi-reflective mirror and a sample chip;
the objective lens is arranged on an optical axis of the beam expanding light and is used for focusing the beam expanding light to form incident light to the upper surface or the lower surface of the cover glass;
the semi-transmitting and semi-reflecting mirror is arranged above the objective lens and is used for transmitting the beam expanding light to the objective lens;
the sample chip is arranged below the objective lens and comprises the cover glass, and the sample chip is used for receiving the incident light of the objective lens and reflecting the incident light to form reflected light;
the detection module comprises a converging mirror and a detector;
the converging mirror is arranged on the optical axis of the reflected light and is used for converging the reflected light to form converging light;
the detector is arranged at a focus behind the converging mirror along the optical axis of the converging light and is used for collecting light spots formed by the converging light;
and the processing unit is used for obtaining the thickness information of the cover glass according to the pixel offset of the light spot and a preset offset relation.
2. The cover slip thickness detection apparatus of claim 1, wherein the transmission module further comprises:
the collimating mirror is arranged on the optical axis of the emitted light and is used for collimating the emitted light to obtain collimated light;
the beam expanding lens is arranged on the optical axis of the collimated light and is used for expanding the collimated light to obtain expanded light;
and the blocking piece is arranged on the optical axis of the beam expanding light and is used for blocking the beam expanding light.
3. The cover glass thickness detection apparatus according to claim 2, wherein the blocking sheet is configured to block the expanded beam light according to a preset blocking rate.
4. The cover glass thickness detection apparatus according to any one of claims 1 to 3, further comprising: and the displacement table is used for loading and moving the sample chip according to a preset stepping amount so as to detect the thickness of the cover glass.
5. A cover glass thickness detection method applied to the cover glass thickness detection apparatus according to any one of claims 1 to 4, the cover glass including an upper surface and a lower surface, the method comprising:
moving the sample chip to a first preset position, acquiring the absolute height of the objective lens when the objective lens and the target surface of the cover glass are focused, and acquiring a first pixel position of the light spot on the detector;
moving the sample chip from the first preset position to a second preset position according to a preset stepping amount to obtain a second pixel position of the light spot on the detector;
selecting a preset offset relationship according to the target surface, wherein the preset offset relationship comprises a first preset offset relationship and a second preset offset relationship;
when the target surface is an upper surface, calculating the defocusing amount of the upper surface of the second preset position according to the first pixel position, the second pixel position and a first preset offset relation, and calculating the measured height of the upper surface according to the defocusing amount of the upper surface and the first absolute height of the objective lens;
when the target surface is a lower surface, calculating the lower surface defocusing amount of a second preset position according to the first pixel position, the second pixel position and a second preset offset relation, and calculating to obtain a lower surface measurement height according to the lower surface defocusing amount and a second absolute height of the objective lens;
and obtaining the thickness of the second preset position of the cover glass according to the upper surface measuring height, the lower surface measuring height and the refractive index.
6. The cover glass thickness detection method according to claim 5, wherein the first pixel position and the second pixel position are pixel positions on the detector at which pixel intensity values are highest at the detection time.
7. The cover slip thickness detection method as claimed in claim 5, further comprising, before said selecting a preset offset relationship based on said target surface:
controlling the objective lens to be focused on the target surface of the cover glass at a preset initial position, and acquiring a first reference pixel position of the light spot on the detector;
controlling the objective lens and the target surface to defocus, recording reference defocus amounts corresponding to N defocus heights, and acquiring a second reference pixel position of the light spot on the detector at each defocus height, wherein N is an integer greater than 1;
calculating a reference pixel offset corresponding to each reference defocus amount, wherein the reference pixel offset is calculated according to the first reference pixel position and the second reference pixel position corresponding to the reference defocus amount;
when the target surface is an upper surface, fitting according to the reference pixel offset and the reference defocus amount to obtain the first preset offset relation;
and when the target surface is a lower surface, fitting according to the reference pixel offset and the reference defocus amount to obtain the second preset offset relationship.
8. The cover glass thickness detection method according to claim 7, wherein the calculating a reference pixel shift amount corresponding to each of the reference defocus amounts, the reference pixel shift amount being calculated from the first reference pixel position and the second reference pixel position corresponding to the reference defocus amount, further comprises:
squaring the difference between the abscissa of the first reference pixel position and the abscissa of the second reference pixel position to obtain a first result;
squaring the difference value of the vertical coordinate of the first reference pixel position and the vertical coordinate of the second reference pixel position to obtain a second result;
adding the first result and the second result and then squaring to obtain a third result;
and multiplying the third result by the pixel size of the detector to obtain the reference pixel offset corresponding to the reference defocus amount.
9. The cover glass thickness detection method according to claim 5, wherein the obtaining the thickness of the second preset position of the cover glass from the upper surface measurement height, the lower surface measurement height and the refractive index further comprises:
acquiring the refractive index of the cover glass;
calculating a measurement height difference according to the upper surface measurement height and the lower surface measurement height;
and obtaining the thickness of the second preset position of the cover glass according to the measured height difference and the refractive index.
10. An electronic device, comprising a memory storing a computer program, and a processor implementing the cover glass thickness detection method according to any one of claims 5 to 9 when the processor executes the computer program.
11. A computer-readable storage medium characterized in that the storage medium stores a program executed by a processor to implement the cover glass thickness detection method according to any one of claims 5 to 9.
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