CN115930829B

CN115930829B - Reconstruction method of confocal microscope

Info

Publication number: CN115930829B
Application number: CN202211685301.3A
Authority: CN
Inventors: 张琥杰; 许陈旭; 张和君; 霍阔
Original assignee: Chotest Technology Inc
Current assignee: Chotest Technology Inc
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-06-23
Anticipated expiration: 2042-12-27
Also published as: CN116878418B; CN116972769B; CN115930829A; CN116878418A; CN116972769A

Abstract

The present disclosure describes a method of confocal microscope reconstruction, comprising: acquiring a plurality of groups of first reflected light beams of an object to be measured, and acquiring a plurality of groups of first measurement information based on the plurality of groups of first reflected light beams; acquiring a plurality of groups of second reflected light beams of the object to be measured, and acquiring a plurality of groups of second measurement information based on the plurality of groups of second reflected light beams; the method comprises the steps of enabling first measurement information comprising a first reflected light beam with the maximum intensity smaller than a first preset value to be first target information, enabling first measurement information comprising the first reflected light beam with the maximum intensity not smaller than the first preset value to be second target information, and enabling second measurement information comprising a second reflected light beam with the maximum intensity smaller than the first preset value and larger than a second preset value to be third target information; and obtaining a target image based on at least two of the first target information, the second target information, and the third target information to reconstruct the object to be measured. According to the present disclosure, a reconstruction method that improves the accuracy of reconstruction of an object to be measured can be provided.

Description

Reconstruction method of confocal microscope

Technical Field

The present disclosure relates to an intelligent manufacturing equipment industry, and in particular to a method for reconstructing a confocal microscope.

Background

At present, optical microscopy is widely applied to various fields of scientific and technical research, industrial measurement and the like, but common optical microscopy (such as a common optical microscope) is difficult to realize three-dimensional morphology reconstruction on an object with a certain thickness. Along with the continuous development of the microscopic technology in recent years, the confocal microscopic technology has become one of important technologies in the field of optical microscopy, has the functional characteristics of high precision, high resolution, non-contact, unique axial tomography imaging and the like, can realize the three-dimensional shape reconstruction of an object to be detected, and is widely applied to the fields of micro-nano detection, precise measurement, life science research and the like.

In general, confocal microscopy is based on the principle that three points of a light source, an object to be measured and a microscope objective are conjugated to each other to mechanically scan to obtain the relative height of the object to be measured, so as to reconstruct the three-dimensional morphology of the object to be measured. Ideally, the relative position of each region to be measured of the object to be measured when the region to be measured is located at the focal plane of the microscope objective can be determined according to the intensity of the reflected light beam of each region to be measured of the object to be measured, so as to determine the relative height of each region to be measured of the object to be measured.

However, for the object to be detected with a non-single structure, the reflectivity distribution of each area to be detected of the object to be detected is inconsistent, when the illumination beam of the light source is stronger under the same measurement condition, the area to be detected with higher reflectivity easily causes the light intensity information of the imaging result to be too high to be distorted (namely, overexposure phenomenon), and the overexposure information generated based on the overexposure phenomenon cannot effectively obtain the relative height of the area to be detected matched with the overexposure information; when the illumination beam of the light source is weak, the reflected beam of the region to be measured having a low reflectance is susceptible to factors such as background noise, resulting in a large error in the relative height of the obtained region to be measured.

Disclosure of Invention

The present disclosure has been made in view of the above-described conventional art, and an object thereof is to provide a method for reconstructing a confocal microscope, which improves the accuracy of measurement of a region to be measured and thus the accuracy of reconstruction of an object to be measured.

To this end, the present disclosure provides a method for reconstructing a confocal microscope, which is applied to a confocal microscope including a microscope objective and reconstructs an object to be measured based on a reflected light beam of the object to be measured, the object to be measured including a plurality of regions to be measured, the method comprising: obtaining reflected light beams of the plurality of areas to be measured as a plurality of groups of first reflected light beams when the object to be measured is illuminated by the first light beams, and obtaining a plurality of groups of first measurement information matched with the plurality of areas to be measured based on the plurality of groups of first reflected light beams, wherein the first measurement information comprises the intensity of the first reflected light beams; obtaining reflected light beams of the plurality of areas to be measured as a plurality of groups of second reflected light beams when the object to be measured is illuminated by the second light beams, and obtaining a plurality of groups of second measurement information matched with the plurality of areas to be measured based on the plurality of groups of second reflected light beams, wherein the intensity of the second light beams is smaller than that of the first light beams, and the second measurement information comprises the intensity of the second reflected light beams; the first measurement information comprising the first reflected light beam with the maximum intensity smaller than a first preset value is made to be first target information, the first measurement information comprising the first reflected light beam with the maximum intensity not smaller than the first preset value is made to be second target information, and the second measurement information comprising the second reflected light beam with the maximum intensity smaller than the first preset value and larger than a second preset value is made to be third target information; and obtaining a target image based on at least two of the first target information, the second target information, and the third target information to reconstruct the object to be measured.

In the present disclosure, by performing multiple measurements on an object to be measured using illumination light beams (first light beam and second light beam) of different intensities, measurement results of the object to be measured under different measurement conditions can be obtained as first measurement information and second measurement information, respectively, and reconstruction accuracy of the object to be measured can be improved by integrating the first measurement information and the second measurement information twice.

In addition, in the reconstruction method related to the present disclosure, optionally, a region to be detected that matches the first target information is a first region, a region to be detected that matches the second target information is a second region, a region to be detected that matches the third target information is a third region, a height of the first region is obtained based on the first target information is a first height, a height of the second region is obtained based on the second target information is a second height, and a height of the third region is obtained based on the third target information is a third height; and obtaining the target image based on at least two of the first height, the second height, and the third height. Therefore, a proper measurement result can be selected automatically according to actual measurement requirements so as to quickly and accurately reconstruct the object to be measured.

In addition, in the reconstruction method related to the present disclosure, optionally, the first measurement information includes first negative defocus information for characterizing an intensity of a first reflected light beam and matching a negative defocus region of the microscope objective, first focus information matching a focus region of the microscope objective, and first positive defocus information matching a positive defocus region of the microscope objective, the first focus information in the first target information is smaller than the first preset value, and the first height is obtained based on the first focus information. In this case, the first target information can be obtained quickly and directly by judging the magnitude relation between the first focusing information and the first preset value, and the first height can be obtained by observing the height corresponding to the occurrence of the first focusing information, whereby the subsequent reconstruction of the object to be measured can be facilitated.

In addition, in the reconstruction method according to the present disclosure, optionally, the first focusing information in the second target information is not smaller than the first preset value, and the second height is obtained based on the first negative defocus information and the first positive defocus region smaller than the first preset value in the second target information. Thereby, a second height can be obtained while maintaining an efficient measurement.

In addition, in the reconstruction method according to the present disclosure, optionally, a first straight line is obtained based on first negative defocus information smaller than the first preset value in the second target information, a second straight line is obtained based on first positive defocus information smaller than the first preset value in the second target information, and the second height is obtained based on the first straight line and the second straight line. Thereby, the second height can be obtained by fitting two straight lines (a first straight line and a second straight line) and based on the two straight lines.

In addition, in the reconstruction method according to the present disclosure, the target image is optionally obtained based on the first height and the second height. In this case, the object to be measured can be reconstructed by a single measurement, whereby the measurement efficiency can be improved.

In addition, in the reconstruction method according to the present disclosure, optionally, the second measurement information includes second negative defocus information that matches a negative defocus region of the microscope objective, second focus information that matches a focus region of the microscope objective, and second positive defocus information that matches a positive defocus region of the microscope objective, and the second focus information in the third target information is smaller than the first preset value and larger than the second preset value, and the third height is obtained based on the second focus region. In this case, the third target information can be obtained by judging whether the second measurement information is smaller than the first preset value and larger than the second preset value, and further the third height can be obtained by observing the height corresponding to the second focusing information of the third target information when the second focusing information appears, whereby the subsequent reconstruction of the object to be measured can be facilitated.

In addition, in the reconstruction method according to the present disclosure, optionally, a first image matching the first region is obtained based on the first height, a third image matching the third region is obtained based on the third height, and the target image is obtained based on the first image and the third image. Under the condition, the object to be measured is illuminated twice by utilizing illumination light beams with different intensities, the intensity of the reflected light beam of the first area and the intensity of the reflected light beam of the third area can be respectively made to be smaller than a first preset value, and then a first image and a third image can be obtained successively, and a target image can be obtained based on the first image and the third image, so that the probability that the intensity of the reflected light beam of the area to be measured is larger than the first preset value due to measurement errors and errors are caused to measurement results can be reduced.

In addition, in the reconstruction method according to the present disclosure, optionally, a region overlapping with the first region and the third region is made to be an overlapping region, a portion of the first image matching the overlapping region is made to be a first overlapping image, a portion of the third image matching the overlapping region is made to be a third overlapping image, and the first image and the third image are aligned and fused based on the first overlapping image and the third overlapping image to obtain the target image. In this case, the first image and the third image can be aligned based on the first superimposed image and the third superimposed image to more accurately obtain the target image, whereby the accuracy of reconstruction of the object to be measured can be improved.

In addition, in the reconstruction method according to the present disclosure, optionally, the first preset value is related to an imaging element that receives the first reflected light beam and the second reflected light beam. In this case, the relationship between the intensity of the reflected light beams (the first reflected light beam and the second reflected light beam) of the region to be measured and the first preset value can be intuitively judged by the imaging element, whereby the first target information, the second target information, and the third target information can be quickly obtained.

According to the reconstruction method of the present disclosure, a confocal microscope reconstruction method can be provided that improves the accuracy of reconstruction of an object to be measured by improving the accuracy of measurement of the area to be measured.

Drawings

The present disclosure will now be explained in further detail by way of example only with reference to the accompanying drawings, in which:

fig. 1 is a schematic diagram illustrating a scenario of a reconstruction system to which examples of the present disclosure relate.

Fig. 2 is a block diagram showing a structure of a measuring apparatus according to an example of the present disclosure.

Fig. 3 is a perspective view showing a certain object to be measured to which the example of the present disclosure relates.

Fig. 4 is a flowchart illustrating a reconstruction method according to an example of the present disclosure.

Fig. 5a is a schematic diagram illustrating a first embodiment of first measurement information related to examples of the present disclosure.

Fig. 5b is a schematic diagram illustrating a second embodiment of the first measurement information to which the examples of the present disclosure relate.

Fig. 6 is a schematic diagram illustrating obtaining a second height in accordance with examples of the present disclosure.

Fig. 7 is a flow chart illustrating a second embodiment of a reconstruction method according to an example of the present disclosure.

Fig. 8 is a schematic diagram showing second measurement information related to an example of the present disclosure.

Fig. 9a is a simplified schematic diagram illustrating a certain analyte according to an example of the present disclosure.

Fig. 9b is a simplified schematic diagram illustrating a first image to which examples of the present disclosure relate.

Fig. 9c is a simplified schematic diagram illustrating a third image to which examples of the present disclosure relate.

Fig. 10 is a flowchart illustrating a third embodiment of a reconstruction method according to an example of the present disclosure.

Detailed Description

Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same members are denoted by the same reference numerals, and overlapping description thereof is omitted. In addition, the drawings are schematic, and the ratio of the sizes of the components to each other, the shapes of the components, and the like may be different from actual ones.

It should be noted that the terms "comprises" and "comprising," and any variations thereof, in this disclosure, such as a process, method, system, article, or apparatus that comprises or has a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or apparatus, but may include or have other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

In addition, headings and the like referred to in the following description of the disclosure are not intended to limit the disclosure or scope thereof, but rather are merely indicative of reading. Such subtitles are not to be understood as being used for segmenting the content of the article, nor should the content under the subtitle be limited only to the scope of the subtitle.

The present disclosure relates to a reconstruction method (hereinafter, may be abbreviated as a reconstruction method or a reconstruction method) of a confocal microscope, which may be used to reconstruct a three-dimensional morphology of an object to be measured. According to the reconstruction method, the same object to be measured is measured for multiple times, so that the situation that the reconstruction of the object to be measured is affected due to measurement errors caused by overexposure can be effectively reduced. This can improve the accuracy of reconstructing the object to be measured.

The reconstruction method of the confocal microscope according to the present disclosure may also be referred to as, for example, a reconstruction method of a microscope, a processing method of an overexposure phenomenon, a reconstruction method with compensation measurement, or the like. It is noted that the names are for illustration of the disclosure, and should not be construed as limiting.

In addition, the present disclosure also provides a reconstruction system (hereinafter may be simply referred to as a reconstruction system) with a confocal microscope, and the reconstruction method related to the present disclosure may be applied to the reconstruction system to reconstruct an object to be measured.

The present disclosure will be described in detail below with reference to the drawings by taking a confocal microscope as an example. It is to be understood that confocal microscopy is only illustrative and the present disclosure is not limited thereto, and any 3D surface topography analyzer for reconstructing an object to be measured can be used to treat overexposure using the methods, systems, or apparatus of the present disclosure.

Fig. 1 is a schematic view illustrating a scenario of a reconstruction system 1 to which examples of the present disclosure relate. Fig. 2 is a block diagram showing a structure of the measurement apparatus 10 according to the example of the present disclosure.

Referring to fig. 1, a reconstruction system 1 of the present disclosure may include a measurement device 10 and a reconstruction device 20. The measuring device 10 may be a confocal microscope. The measurement device 10 may be configured to measure the object 2 to obtain measurement information that can reflect the object 2, and the reconstruction device 20 may be configured to receive the measurement information and process the measurement information to reconstruct the object 2. Thereby, the measurement of the object 2 to be measured can be realized and the reconstruction of the object 2 to be measured can be completed based on the measurement information. In the present disclosure, reconstructing the object 2 may refer to reconstructing a three-dimensional morphology of the object 2.

In some examples, analyte 2 may be referred to as a sample. The sample can be a semiconductor, a 3C electronic glass screen, a micro-nano material, an automobile part, or an ultra-precise device such as a MEMS device. In some examples, the sample may be a device that is used in the field of aerospace, and the like. In other examples, the sample may be a tissue or cell slice of a biological domain. However, the present disclosure is not limited thereto, and the object 2 to be measured may be any device requiring precise measurement.

Referring to fig. 1, in some examples, the measurement device 10 may include a load bearing platform 110. The carrying platform 110 may be used to carry the object 2 to be measured. In some examples, the measurement device 10 may include a microscope objective 120. The micro objective 120 may be used to scan the object 2 to be measured. This enables measurement of the object 2 to be measured.

In some examples, the reconstruction method may be a method applied to a confocal microscope. In some examples, the reconstruction method may reconstruct the object 2 based on the reflected light beam of the object 2.

In some examples, the measurement device 10 may include an illumination module 130 and an adjustment module 140 (see fig. 2). The illumination module 130 may be configured to emit an illumination beam (e.g., a first beam and/or a second beam, hereinafter), and the adjustment module 140 may be configured to adjust a propagation path of the illumination beam emitted by the illumination module 130 so that the illumination beam reaches the surface of the object 2 to be measured. In some examples, the illumination beam may be reflected by the object 2 into a reflected beam after reaching the object 2.

In some examples, referring to fig. 2, the measurement device 10 may further include an imaging module 150. The imaging module 150 may be configured to receive a reflected light beam reflected by the object 2 to be measured.

Fig. 3 is a perspective view showing a certain object 2 to be measured according to an example of the present disclosure.

In some examples, the test object 2 may include a plurality of regions to be tested. It should be noted that a region of the present disclosure having the same characteristics may refer to one region to be measured. In other words, each region under test may have different characteristics. In some examples, portions having the same flatness may be used as one area to be measured. For example, referring to fig. 3, if the plane P1 and the plane P2 have the same flatness, the plane P1 and the plane P2 may be used as one area to be measured; if the inclined planes P3 and P4 have the same inclination, the inclined planes P3 and P4 may serve as one region to be measured.

In other examples, portions having the same reflectivity may be used as one area to be measured. For example, referring to fig. 3, if the plane P1 and the plane P2 have the same reflectivity, the plane P1 and the plane P2 may be used as one area to be measured; if the inclined plane P3 and the inclined plane P4 have the same reflectivity, the inclined plane P3 and the inclined plane P4 may serve as one area to be measured. In this case, the object 2 to be measured having a non-single structure can be divided into a plurality of areas to be measured according to the preset characteristics, and further, the subsequent reconstruction of the object 2 to be measured can be facilitated.

The reconstruction method according to the present disclosure will be specifically described below by taking the example in which the object 2 includes at least two regions to be measured, but the present disclosure is not limited thereto. It is understood that the same effects as the present disclosure can be achieved by repeating part of the steps in the present disclosure for the object 2 including more regions to be measured.

Fig. 4 is a flowchart illustrating a first embodiment of a reconstruction method according to an example of the present disclosure.

In some examples, referring to fig. 4, the reconstruction method may include: a plurality of sets of first reflected light beams of the object 2 to be measured at the time of the first light beam illumination are acquired, a plurality of sets of first measurement information are acquired based on the plurality of sets of first reflected light beams (step S100), first target information and second target information are acquired based on the plurality of sets of first measurement information (step S300), and the object 2 to be measured is reconstructed based on the first target information and the second target information (step S500).

In step S100, a plurality of sets of first reflected light beams of the object 2 to be measured at the time of the first light beam illumination may be acquired. In some examples, the object 2 to be measured may be measured under illumination conditions when the first light beams are illuminated to obtain a plurality of sets of first reflected light beams. In some examples, the plurality of sets of first reflected light beams may be light beams that are one-to-one matched to the plurality of areas under test. In other words, the reflected light beams of the plurality of regions to be measured of the object to be measured 2 at the time of the illumination of the first light beam can be acquired as the plurality of sets of first reflected light beams. For example, if the object 2 includes two regions to be measured, two sets of first reflected light beams of the object 2 when the first light beams illuminate can be obtained, and each set of first reflected light beams is matched with each region to be measured one by one. In this case, each region to be measured can be reconstructed based on each set of first reflected light beams that match each region to be measured.

In some examples, the intensity of the first light beam may be related to a lower reflectivity region to be measured in each region to be measured. In some examples, the object 2 may have at least one region to be measured that is not overexposed when illuminated by the first light beam. Thereby, at least one region to be measured can be reconstructed based on the reflected light beam of the region to be measured where the overexposure phenomenon does not occur.

In some examples, step S100 may further include obtaining a plurality of sets of first measurement information based on the plurality of sets of first reflected light beams. In some examples, the plurality of sets of first measurement information may be matched to the plurality of regions under test. In this case, the plurality of regions to be measured can be reconstructed based on the plurality of sets of first measurement information that match the plurality of regions to be measured.

Fig. 5a is a schematic diagram illustrating a first embodiment of first measurement information related to examples of the present disclosure. Fig. 5b is a schematic diagram illustrating a second embodiment of the first measurement information to which the examples of the present disclosure relate. Wherein fig. 5a shows first measurement information matched with a region to be measured where the exposure phenomenon does not occur, and fig. 5b shows first measurement information matched with a region to be measured where the exposure phenomenon occurs. In the present disclosure, the first measurement information shown in fig. 5a may correspond to a region to be measured with a low reflectivity, and the first measurement information shown in fig. 5b may correspond to a region to be measured with a high reflectivity.

In some examples, the first measurement information may include an intensity of the first reflected light beam. In some examples, the heights of the respective regions to be measured may be obtained based on the intensities of the sets of first reflected light beams. This can reconstruct a plurality of regions to be measured based on the heights of the respective regions to be measured, thereby reconstructing the object to be measured 2.

In some examples, when measuring the object 2, the micro objective 120 may perform a longitudinal scan of the object 2 to achieve measurement of each region to be measured of the object 2. Specifically, in some examples, during measurement of the object 2, the object 2 may be located in the negative defocus region, the focus region, and the positive defocus region of the microscope objective 120 in order; in some examples, during measurement of object 2, object 2 may be located in the positive defocus region, the focus region, and the negative defocus region of microscope objective 120 in this order. This enables measurement of the object 2 to be measured.

In some examples, the height of the object 2 may be obtained based on the reflected light beam when the object 2 is located in the focusing region of the microscope objective 120.

Referring to fig. 5a, in some examples, the first measurement information may include first negative defocus information characterizing the intensity of the first reflected beam (vertical axis in fig. 5 a) and matching the negative defocus region of the microscope objective 120, first focus information matching the focus region of the microscope objective 120, and first positive defocus information matching the positive defocus region of the microscope objective 120. The first focusing information refers to information corresponding to B1, the first negative defocus information refers to information located on the left side of B1, and the first positive defocus information refers to information located on the right side of B1. In this case, the first negative defocus information, the first focus information, and the first positive defocus information can be integrated to more accurately obtain the heights of the respective regions to be measured, and thus the reconstruction accuracy of the respective regions to be measured can be improved. In some examples, the first focusing information may reflect a maximum intensity of the first reflected light beam.

As described above, the measurement device 10 may include an imaging module 150. In some examples, imaging module 150 may include imaging elements for converting optical signals into electrical signals. In particular, the imaging element may receive the first reflected light beam. In some examples, the imaging element may receive the first reflected light beam and convert the first reflected light beam into an electrical signal. In some examples, the imaging element may be further configured to convert the first reflected light beam into an electrical signal and then convert the electrical signal into a digital signal according to a predetermined coefficient.

In some examples, a digital signal obtained based on the first reflected light beam may be used to characterize the intensity of the first reflected light beam. In some examples, the digital signal used to reflect the intensity of the first reflected beam may be a gray value. That is, the intensity of the first reflected light beam included in the first measurement information may be represented by a gray value.

Referring to fig. 5a, in some examples, the first measurement information may also include a height of the area under test (horizontal axis in fig. 5 a) that matches the first reflected beam. In some examples, the measurement device 10 may also include a location recording module. In some examples, the height of the area under test may be obtained based on readings of a location recording module. In some examples, the position recording module may be a grating ruler, a PTZ (piezoceramic) or the like.

Generally, the intensity range of the light signal received by the imaging element may be referred to as a dynamic range, and if the intensity of the reflected light beam exceeds the dynamic range, the imaging element cannot accurately present the intensity of the reflected light beam.

In some examples, for a region to be measured with higher reflectivity, if the intensity of its reflected beam exceeds the dynamic range of the imaging element, the first measurement information matched thereto may be as shown in fig. 5 b. It can be seen from fig. 5b that the first measurement information has a tendency to "flat top" for the region to be measured where the reflectivity is high, due to the limitation of the dynamic range of the imaging element. That is, for reflected beams having intensities outside the dynamic range of the imaging element, the imaging element reflects at its upper limit of dynamic range.

In some examples, when the region to be measured is overexposed, the first measurement information matched with the region to be measured cannot be fully embodied. For example, fig. 5b shows a portion of the first measurement information that matches the negative defocus region of the microscope objective 120 and a portion of the first positive defocus information that matches the positive defocus region of the microscope objective 120. This is because the closer the area to be measured is to the focus area of the microscope objective 120, the stronger the intensity of the reflected beam of the area to be measured, which will result in the larger the first negative defocus information close to the first focus information and the first positive defocus information close to the first focus information, which are each presented in fig. 5b as the upper limit of the dynamic range of the imaging element when the first negative defocus information, the first focus information, and the first positive defocus information exceed the dynamic range of the imaging element.

As described above, the reconstruction method may include step S300, and in step S300, the first target information and the second target information may be acquired based on the plurality of sets of first measurement information. As described above, a plurality of sets of first measurement information can be obtained based on a plurality of sets of first reflected light beams. In some examples, the first preset value may be as shown by I1 in fig. 5a and 5 b.

In some examples, the first measurement information that is less than the first preset value may be made the first target information. In the present disclosure, when the first focusing information in the first measurement information is smaller than a first preset value, the first measurement information is considered to be smaller than the first preset value; when the first focusing information in the first measurement information is not smaller than the first preset value, the first measurement information is considered to be not smaller than the first preset value. That is, in some examples, the first measurement information including the intensity of the first reflected light beam smaller than the first preset value may be made the first target information. In other words, the first measurement information including the first reflected light beam having the maximum intensity smaller than the first preset value may be made the first target information (for example, the first measurement information shown in fig. 5 a). Thus, the first target information can be obtained by determining whether the intensity of the first reflected light beam is smaller than the first preset value.

In some examples, the intensity of the first reflected beam of the region to be measured with the lowest reflectivity may be greater than the background noise under measurement conditions when the first beam is illuminated. Thus, the object 2 to be measured can be measured completely and accurately.

In some examples, the first measurement information that is not less than the first preset value may be made the second target information. That is, the first measurement information including the intensity of the first reflected light beam not smaller than the first preset value may be made the second target information. In other words, in some examples, the first measurement information including the maximum intensity of the first reflected light beam not less than the first preset value may be made the second target information (e.g., the first measurement information shown in fig. 5 b). Thus, the second target information can be obtained by determining whether or not the intensity of the first reflected light beam is not less than the first preset value.

In some examples, the first focus information in the first target information may be less than a first preset value. In some examples, the first focusing information in the second target information may be not less than the first preset value. Thus, the first target information and the second target information can be obtained quickly and directly by judging the relationship between the first focusing information and the first preset value.

In some examples, the first preset value may be associated with an imaging element that receives the first reflected light beam and the second reflected light beam. Preferably, it may be related to the dynamic range of the imaging element. In this case, the relationship between the intensity of the reflected light beams (the first reflected light beam and the second reflected light beam) of the region to be measured and the first preset value can be intuitively judged by the imaging element, whereby the first target information, the second target information, and the third target information (described later) can be obtained quickly.

In some examples, the first preset value may not be greater than a dynamic range of the imaging element. Assuming that the dynamic range of the imaging element is 0 to 255, the first preset value may be 255 in an ideal case. However, in the actual measurement process, considering the influence of environmental factors and the like, a value different from 255 by a predetermined value may be required as the first preset value according to the actual measurement requirement, for example, the first preset value may be 230, 240, 245, 250, and the like. However, the present disclosure is not limited thereto, and the first preset value may be set to any value according to the dynamic range of the imaging element and the actual measurement requirement.

As described above, in some examples, the reconstruction method may further include reconstructing the object 2 to be measured based on the first target information and the second target information (step S500).

In some examples, the region to be measured that matches the first target information may be a first region, the region to be measured that matches the second target information may be a second region, the height of the first region may be obtained based on the first target information as a first height, and the height of the second region may be obtained based on the second target information as a second height. In some examples, the target image may be obtained based on the first height and the second height. In this case, the object 2 to be measured can be reconstructed by a single measurement, whereby the measurement efficiency can be improved. In some examples, a target image may be obtained to reconstruct the test object 2.

Referring to fig. 5a, in some examples, a first height may be obtained based on the first focus information (e.g., Z1 in fig. 5a may represent the first height). In this case, for the region to be measured (i.e., the first region) where the exposure phenomenon does not occur, the first height can be obtained by observing the height corresponding to the occurrence of the first focusing information, whereby the subsequent reconstruction of the object to be measured 2 can be facilitated.

Due to the limitation of the dynamic range of the imaging element, the first focusing information in the second target information can only be presented in the measurement result with the upper limit of the dynamic range, no matter how large it is. In other words, the first focusing information in the second target information cannot be presented in the measurement result with its true value. Thus, in some examples, the second height may be obtained based on the first negative defocus information and the first positive defocus region that are less than the first preset value in the second target information. Thereby, a second height can be obtained while maintaining an efficient measurement.

Referring to fig. 6, in some examples, a first straight line L1 may be obtained based on first negative defocus information smaller than a first preset value in the second target information, a second straight line L2 may be obtained based on first positive defocus information smaller than the first preset value in the second target information, and a second height may be obtained based on the first straight line L1 and the second straight line L2. Thus, the second height can be obtained by fitting two straight lines (the first straight line L1 and the second straight line L2) and based on the two straight lines.

In some examples, the accuracy of the measurement information may be higher for regions to be measured that have higher reflectivity, the farther away from the focal region of the microscope objective 120. Therefore, a second height of higher accuracy can be obtained based on the first negative defocus information and the first positive defocus information distant from the first focus information for fitting two straight lines.

In some examples, the second height may be obtained based on an intersection of the first line L1 and the second line L2. Specifically, in some examples, the intersection B2 of the first straight line L1 and the second straight line L2 may be considered as first focus information, and the height corresponding to the first focus information may be a second height (e.g., Z2 in fig. 6 may represent the second height). In this case, since the measurement error of the first negative defocus information for fitting the first straight line L1 and the positive defocus information for fitting the second straight line L2 is small, the second height obtained based on the first straight line L1 and the second straight line L2 can more accurately reflect the relative height of the second region.

In some examples, for the first target information, two straight lines may also be fitted based on the first negative defocus information and the first positive defocus information distant from the first focus information to obtain the first height. In this case, the measurement accuracy of the first region can be improved, and further, the reconstruction accuracy of the subsequent object 2 to be measured can be improved.

It is to be understood that the first height of the present disclosure corresponds to the heights of all points of the first region (which may or may not be the same) and should not be construed as a single value. The second height and the third height are the same.

In some examples, referring to fig. 7, the reconstruction method may include: acquiring a plurality of groups of first reflected light beams of the object 2 to be measured when the first light beams are illuminated, acquiring a plurality of groups of first measurement information based on the plurality of groups of first reflected light beams (step S200), acquiring a plurality of groups of second reflected light beams of the object 2 to be measured when the second light beams are illuminated, acquiring a plurality of groups of second measurement information based on the plurality of groups of second reflected light beams (step S400), acquiring first target information based on the plurality of groups of first reflected light beams, acquiring third target information based on the plurality of groups of second reflected light beams (step S600), and reconstructing the object 2 to be measured based on the first target information and the third target information (step S800). In this case, the object 2 to be measured is measured multiple times by using different illumination light beams, so that reflected light beams of the object 2 to be measured under different illumination light beams can be obtained, and further, the object 2 to be measured can be reconstructed by combining different reflected light beams, so as to improve the reconstruction accuracy of the object 2 to be measured.

In some examples, step S200 may be the same as the acquisition of the plurality of sets of first measurement information in step S100 in the first embodiment of the above-mentioned reconstruction method, and will not be described herein.

In some examples, in step S400, a plurality of sets of second reflected light beams of the object 2 to be measured at the time of illumination of the second light beams may be acquired. In some examples, the object 2 to be measured may be measured under illumination conditions when the second light beam is illuminated to obtain a plurality of sets of second reflected light beams. In some examples, the plurality of sets of second reflected light beams may be light beams that match the plurality of regions under test. In other words, the reflected light beams of the plurality of regions to be measured of the object to be measured 2 at the time of illumination of the second light beam can be acquired as the plurality of sets of second reflected light beams. It should be noted that the plurality of regions to be measured in step S400 and the plurality of regions to be measured in step S100 may be the same. Thereby, each region to be measured can be reconstructed based on the second reflected light beam matched with each region to be measured.

In some examples, the second light beam may be obtained by adjusting the intensity of the illumination light beam emitted by the illumination module 130. In some examples, the intensity of the second light beam may be less than the intensity of the first light beam. In this case, the possibility of overexposure in the region to be measured having a high reflectance can be reduced.

In some examples, the intensity of the second light beam may be determined by adjusting the illumination module 130 and observing the state of the imaging element, when the intensity of each set of reflected light beams received by the imaging element is observed not to exceed the dynamic range, then the intensity of the second light beam may be determined. In some examples, the state of the imaging element may be observed by means of external software or devices. Thereby, it can be facilitated to determine the intensity of the second light beam.

In some examples, the object 2 may not have the overexposure phenomenon in the region to be measured under the measurement condition when the second light beam is illuminated. In this case, the imaging element is able to receive and present the sets of measurement information that are able to reflect the intensity of the second reflected light beam in its entirety.

In some examples, step S400 may further include obtaining a plurality of sets of second measurement information based on the plurality of sets of second reflected light beams. In some examples, the plurality of sets of second measurement information may be matched to the plurality of regions under test. In this case, the plurality of regions to be measured can be reconstructed based on the plurality of sets of first measurement information that match the plurality of regions to be measured. In some examples, the first reflected beam and the second reflected beam of the area under test may have a higher signal-to-noise ratio. Thus, the measurement accuracy of the region to be measured can be improved.

In some examples, the second measurement information may include an intensity (vertical axis in fig. 8) of the second reflected light beam. In some examples, the heights of the respective regions to be measured may be obtained based on the intensities of the sets of second reflected light beams. This can reconstruct a plurality of regions to be measured based on the heights of the respective regions to be measured, thereby reconstructing the object to be measured 2. In some examples, the second measurement information may also include a height of the area under test (horizontal axis Z in fig. 8) that matches the second reflected beam.

For a clearer illustration of the present disclosure, fig. 8 schematically integrates the second measurement information of two areas to be measured into one graph, and the second measurement information of higher intensity in fig. 8 may correspond to the first measurement information in fig. 5b, and the second measurement information of lower intensity may correspond to a part of the first measurement information in fig. 5 a. The correspondence here may refer to correspondence with the same region to be measured.

Referring to fig. 8, in some examples, the second measurement information may include second negative defocus information that matches a negative defocus region of the microscope objective 120, second focus information that matches a focus region of the microscope objective 120, and second positive defocus information that matches a positive defocus region of the microscope objective 120. The second focusing information refers to information corresponding to B3, the first negative defocus information refers to information located on the left side of B3, and the first positive defocus information refers to information located on the right side of B3. In this case, the second negative defocus information, the second focus information, and the second positive defocus information can be integrated to more accurately obtain the heights of the respective regions to be measured, and thus the reconstruction accuracy of the respective regions to be measured can be improved. In some examples, the second focus information may reflect a maximum intensity of the second reflected light beam.

In some examples, in step S600, first target information may be acquired based on a plurality of sets of first reflected light beams. In some examples, the first target information acquired in step S600 may be the same as the first target information acquired in step S300 in the first embodiment, and will not be described herein. In some examples, acquiring the first target information based on the plurality of sets of first reflected light beams may be included in step S200.

In some examples, step S600 may further include obtaining third target information based on the plurality of sets of second reflected light beams. In some examples, obtaining third target information based on the plurality of sets of second reflected light beams may be included in step S400. As described above, a plurality of sets of second measurement information can be obtained based on a plurality of sets of second reflected light beams. In some examples, the second measurement information smaller than the first preset value may be made the third target information. In some examples, the third target information may also be greater than the second preset value. In some examples, the second measurement information that is smaller than the first preset value and larger than the second preset value may be made the third target information. That is, the second measurement information including the intensity of the second reflected light beam smaller than the first preset value and larger than the second preset value may be made the third target information. In other words, the second measurement information including the second reflected light beam having the maximum intensity smaller than the first preset value and larger than the second preset value may be made the third target information, for example, it may be seen from fig. 8 that the second measurement information near the origin of coordinates on the left side does not belong to the third target information and the second measurement information far from the origin of coordinates on the right side belongs to the third target information. In this case, the third target information can be conveniently and accurately obtained by judging whether the second measurement information is smaller than the first preset value and larger than the second preset value.

In some examples, the second focusing information in the third target information may be smaller than the first preset value and larger than the second preset value. Thus, the third target information can be obtained quickly and directly by judging the relation between the second focusing information and the first preset value and the second preset value.

In some examples, the second preset value may be related to the measurement environment of the object 2 to be measured. In some examples, the second preset value may be background noise. In some examples, the second preset value may be set to a value greater than background noise according to actual measurement requirements.

As described above, the reconstruction method may further include step S800, and in step S800, the object 2 to be measured may be reconstructed based on the first target information and the third target information.

Fig. 9a is a simplified schematic diagram illustrating a certain analyte 2 according to an example of the present disclosure. Fig. 9b is a simplified schematic diagram illustrating a first image to which examples of the present disclosure relate. Fig. 9c is a simplified schematic diagram illustrating a third image to which examples of the present disclosure relate.

As described above, the first height may be obtained based on the first target information. In some examples, a third altitude may be obtained based on the third target information. And enabling the region to be detected, which is matched with the third target information, to be a third region, and enabling the height of the third region to be obtained based on the third target information to be used as a third height.

In some examples, the third height may be obtained based on the second focus information (e.g., Z3 in fig. 8 may represent the third height). In this case, the third height can be obtained by observing the height corresponding to the second focusing information of the third target information when it appears, and thus, the subsequent reconstruction of the object 2 to be measured can be facilitated.

In some examples, for the object 2 to be measured with a relatively complex structure, there may be a case where the first measurement information of the area of the part of the object 2 to be measured belongs to the first target information and the second measurement information belongs to the third target information. In other words, there is a part of the area to be measured which is not exposed when the first light beam is illuminated; the exposure phenomenon also occurs when the second light beam is illuminated, and meanwhile, the intensity of the reflected light beam is not lower than a second preset value. In this case, since the first target information matches the first region and the third target information matches the third region, the first region and the third region may exist in partially the same region, that is, the overlapping region.

Referring to fig. 9a, in some examples, the object 2 may include three regions to be measured, namely, a region to be measured Q1, a region to be measured Q2, and a region to be measured Q3, where the region to be measured (i.e., the region to be measured where the exposure phenomenon does not occur when the first beam is illuminated, also referred to as a first region Q12) that is matched with the first target information is the region to be measured Q1 and the region to be measured Q2, and the region to be measured (also referred to as a third region Q23) that is matched with the third target information is the region to be measured Q2 and the region to be measured Q3, and then the region to be measured Q2 may be a coincident region.

In some examples, the target image may be obtained based on the first height and the third height. In some examples, a first image matching the first region may be obtained based on the first height, e.g., see fig. 9b, and a first image P12 matching the first region Q12 may be as shown. In some examples, a third image P23 matching the third region Q23 may be obtained based on the third height, for example, see fig. 9c, and the third image P23 matching the third region Q23 may be as shown in fig. 9 c.

In some examples, the target image may be obtained based on the first image and the third image. In some examples, the target image may be an image that matches the object 2 to be measured. In this case, the object 2 to be measured is illuminated and measured twice by using illumination light beams with different intensities, so that the intensity of the reflected light beam in the first area and the intensity of the reflected light beam in the third area can be respectively made smaller than a first preset value, and further, a first image and a third image can be obtained successively, and a target image can be obtained based on the first image and the third image, thereby, the probability that the intensity of the reflected light beam in the area to be measured is larger than the first preset value due to measurement errors and errors occur in measurement results can be reduced.

As described above, the first region and the third region may have overlapping regions. In some examples, it may be determined whether there is a coincident region of the first region and the third region based on the first target information and the third target information. In some examples, it may be determined whether there is a coincident region of the first region and the third region based on the first image and the third image.

In some examples, the overlapping region of the first region and the third region is made to be an overlapping region (for example, the region to be measured Q2 in fig. 9 a), the portion of the first image that matches the overlapping region is made to be a first overlapping image (for example, the first overlapping image Q21 in fig. 9 b), the portion of the third image that matches the overlapping region is made to be a third overlapping image (for example, the third overlapping image Q22 in fig. 9 c), and the first image and the third image may be aligned and fused based on the first overlapping image and the third overlapping image to obtain the target image. In this case, the first image and the third image can be aligned based on the first superimposed image and the third superimposed image to more accurately obtain the target image, whereby the reconstruction accuracy of the object 2 to be measured can be improved.

And the height obtained by the first target information matched with the overlapping area is the first overlapping height, and the height obtained by the third target information matched with the overlapping area is the third overlapping height. In some examples, in the target image, the height of the overlapping region may be an average of the first overlapping height and the third overlapping height. In this case, the accuracy of the overall measurement of the object 2 to be measured can be improved by averaging the measurement results of a plurality of times, and the accuracy of the reconstruction of the object 2 to be measured can be improved.

Referring to fig. 10, in the present embodiment, the reconstruction method may include acquiring a plurality of sets of first reflected light beams of the object 2 at the time of first light beam illumination (step S910), acquiring first target information and second target information based on the plurality of sets of first reflected light beams (step S930), acquiring a plurality of sets of second reflected light beams of the object 2 at the time of second light beam illumination (step S950), acquiring third target information based on the plurality of sets of second reflected light beams (step S970), and acquiring a target image based on at least two of the first target information, the second target information, and the third target information to reconstruct the object 2 (step S990).

In some examples, the contents of steps S910, S930, S950, S970 may be the same as those of the above two embodiments, and will not be described herein.

As described above, the first height may be obtained based on the first target information, the second height may be obtained based on the second target information, and the third height may be obtained based on the third target information. In some examples, the target image may be obtained based on at least two of the first height, the second height, and the third height. Therefore, a proper measurement result can be selected independently according to actual measurement requirements so as to quickly and accurately reconstruct the object 2 to be measured.

In other examples, when the step S990 obtains the target image based on the first height, the second height, and the third height, the target image obtained in the step S500 in the first embodiment and the target image obtained in the step S800 in the second embodiment may be fused. Thereby, the reconstruction accuracy can be improved.

In addition, the present disclosure also provides a reconstruction device, which may be used to implement the above reconstruction method. In some examples, the reconstruction apparatus may include a receiving module, a screening module, and a processing module. Wherein the individual modules can be connected by signals.

In some examples, the receiving module may be configured to receive first measurement information and/or second measurement information obtained by measurement by the measurement device. In some examples, the screening module may screen the first target information and the second target information from the first measurement information according to a preset first preset value. In some examples, the screening module may screen first target information from the first measurement information and screen third target information from the second measurement information. In some examples, the processing module may reconstruct the object 2 based on the received first target information and second target information. In some examples, the processing module may reconstruct the object 2 based on the received first target information and third target information. In some examples, the processing module may reconstruct the object 2 to be measured based on the received first target information, second target information, and third target information.

It will be appreciated that the present disclosure is not limited to the number of adjustment times of the illumination beam, and in some examples, for an object 2 to be measured with a more complex structure, the intensity of the illumination beam may be adjusted multiple times to measure the object 2 multiple times until each area to be measured of the object 2 is not exposed on the premise that the reflected beam of each area to be measured can be maintained to have a higher signal-to-noise ratio at least once. This can improve the accuracy of reconstructing the object 2.

In the present disclosure, by performing multiple measurements on the object 2 to be measured using illumination light beams (first light beam and second light beam) of different intensities, measurement results of the object 2 to be measured under different measurement conditions can be obtained as first measurement information and second measurement information, respectively, and reconstruction accuracy of the object 2 to be measured can be improved by integrating the first measurement information and the second measurement information twice.

While the disclosure has been described in detail in connection with the drawings and examples, it is to be understood that the foregoing description is not intended to limit the disclosure in any way. Modifications and variations of the present disclosure may be made as desired by those skilled in the art without departing from the true spirit and scope of the disclosure, and such modifications and variations fall within the scope of the disclosure.

Claims

1. A method for reconstructing a confocal microscope, which is applied to a confocal microscope including a microscope objective and based on a reflected light beam of an object to be measured to reconstruct the object to be measured, the object to be measured including a plurality of regions to be measured, the method comprising: obtaining reflected light beams of the plurality of areas to be measured as a plurality of groups of first reflected light beams when the object to be measured is illuminated by the first light beams, and obtaining a plurality of groups of first measurement information matched with the plurality of areas to be measured based on the plurality of groups of first reflected light beams, wherein the first measurement information comprises the intensity of the first reflected light beams; obtaining reflected light beams of the plurality of areas to be measured as a plurality of groups of second reflected light beams when the object to be measured is illuminated by the second light beams, and obtaining a plurality of groups of second measurement information matched with the plurality of areas to be measured based on the plurality of groups of second reflected light beams, wherein the intensity of the second light beams is smaller than that of the first light beams, and the second measurement information comprises the intensity of the second reflected light beams; the first measurement information comprising the first reflected light beam with the maximum intensity smaller than a first preset value is made to be first target information, the first measurement information comprising the first reflected light beam with the maximum intensity not smaller than the first preset value is made to be second target information, and the second measurement information comprising the second reflected light beam with the maximum intensity smaller than the first preset value and larger than a second preset value is made to be third target information; the method comprises the steps of enabling a region to be detected, which is matched with first target information, to be a first region, enabling a region to be detected, which is matched with second target information, to be a second region, enabling a region to be detected, which is matched with third target information, to be a third region, enabling the height of the first region to be obtained based on the first target information to be used as a first height, enabling the height of the second region to be obtained based on the second target information to be used as a second height, and enabling the height of the third region to be obtained based on the third target information to be used as a third height; and obtaining a target image based on at least two of the first height, the second height, and the third height to reconstruct the object under test;

Wherein the first measurement information includes first negative defocus information for characterizing an intensity of a first reflected light beam and matching a negative defocus region of the microscope objective, first focus information matching a focus region of the microscope objective, and first positive defocus information matching a positive defocus region of the microscope objective, first focus information in the first target information being smaller than the first preset value, the first height being obtained based on the first focus information; the first focusing information in the second target information is not smaller than the first preset value, the second height is obtained based on first negative defocus information and first positive defocus regions smaller than the first preset value in the second target information, a first straight line is obtained based on first negative defocus information smaller than the first preset value in the second target information, a second straight line is obtained based on first positive defocus information smaller than the first preset value in the second target information, and the second height is obtained based on the first straight line and the second straight line; the second measurement information comprises second negative defocus information matched with a negative defocus region of the microscope objective, second focus information matched with a focus region of the microscope objective, and second positive defocus information matched with a positive defocus region of the microscope objective, the second focus information in the third target information is smaller than the first preset value and larger than the second preset value, and the third height is obtained based on the second focus information.

2. The method of confocal microscopy reconstruction of claim 1, wherein:

the target image is obtained based on the first height and the second height.

3. The method of confocal microscopy reconstruction of claim 1, wherein:

a first image matching the first region is obtained based on the first height, a third image matching the third region is obtained based on the third height, and the target image is obtained based on the first image and the third image.

4. The confocal microscope reconstruction method of claim 3 wherein:

and enabling the overlapped region in the first region and the third region to be an overlapped region, enabling the part matched with the overlapped region in the first image to be a first overlapped image, enabling the part matched with the overlapped region in the third image to be a third overlapped image, aligning and fusing the first image and the third image based on the first overlapped image and the third overlapped image to obtain the target image.

5. The method of confocal microscopy reconstruction of claim 1, wherein:

The first preset value is associated with an imaging element that receives the first reflected light beam and the second reflected light beam.