CN110763154A - Large-field-of-view microscopic 3D (three-dimensional) morphology multi-channel measuring device and method - Google Patents
Large-field-of-view microscopic 3D (three-dimensional) morphology multi-channel measuring device and method Download PDFInfo
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Abstract
The invention relates to a large-field microscopic 3D appearance multi-channel measuring device and a method, wherein the device comprises: the device comprises a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module, a multi-channel spectrum gray level image acquisition module, a high-precision three-axis objective table and an image analysis and objective table control module. The device can realize automatic focusing once and complete the measurement of the microscopic 3D shape of the sample in a large view field under the condition of no axial movement of the object carrying table. The method can realize microscopic 3D shape measurement with high transverse resolution, high longitudinal measurement precision, millimeter-scale longitudinal measurement range and inch-scale large-field transverse measurement range.
Description
Technical Field
The invention belongs to the field of optical microscopic imaging, and relates to a large-field microscopic 3D morphology measuring device and method.
Background
The existing microscopic three-dimensional shape measurement technology has a plurality of defects, such as small observation range of scanning type microscopic detection and low environment anti-interference capability; the interferometric method requires a large number of axial scans, which limits the measurement efficiency of the method; the traditional confocal microscopic measurement single-point mechanical scanning is difficult to realize real-time and rapid three-dimensional measurement; although the parallel confocal measurement technology realizes simultaneous detection of sampling points on the same confocal section, longitudinal scanning or mechanical scanning of auxiliary equipment is still required, and the measurement efficiency and the measurement accuracy are limited to a certain extent by starting and stopping the scanning process for many times and vibration of the mechanical scanning. For example, the conventional application number cn201510922156.x solves the problem of layer-by-layer longitudinal scanning of the conventional parallel confocal measurement technology, but still needs to acquire sample gray level images before and after the focal plane of a local observation field, mechanical start-stop motion exists, the efficiency is low, and the expansion of microscopic 3D morphology restoration from the local observation field to a large field is limited to a certain extent. Therefore, there is a need for an optical measurement method to solve the above problems to achieve high precision, high efficiency, and large field range of microscopic 3D topography.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a large-view-field microscopic 3D appearance multi-channel measuring device and a large-view-field microscopic 3D appearance multi-channel measuring method. The device and the large-view-field microscopic 3D morphology multi-channel measuring method implemented on the device can realize the large-view-field microscopic 3D morphology measurement of the sample without axial movement of the object carrying table after primary automatic focusing. The method can realize microscopic 3D shape measurement with high transverse resolution, high longitudinal measurement precision, millimeter-scale longitudinal measurement range and inch-scale large-field transverse measurement range.
The technical scheme of the invention is as follows:
the large-field-of-view microscopic 3D morphology multi-channel measuring device comprises a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module and an image analysis and objective table control module.
The compound color light illumination module is sequentially provided with the following components in the light path propagation direction: the device comprises a polychromatic light source, a condenser, a uniform collimating light lens group, a semi-reflecting and semi-transmitting spectroscope and an objective lens with longitudinal dispersion.
The longitudinal dispersion enhanced optical imaging module is sequentially provided with the following components in the light path propagation direction: the device comprises a high-precision three-axis objective table, an objective lens with longitudinal dispersion, a semi-reflecting semi-permeable spectroscope, a tube lens with longitudinal dispersion and a multi-channel spectrum gray image acquisition module.
The image analysis and objective table control module is sequentially provided with the following components in a signal transmission direction: the system comprises a multi-channel spectrum gray level image acquisition module, an image analysis and objective table control system and a high-precision three-axis objective table.
The polychromatic light illumination module and the longitudinal dispersion enhanced optical imaging module share a semi-reflecting semi-transparent spectroscope and an objective lens with longitudinal dispersion.
The longitudinal dispersion enhanced optical imaging module and the image analysis and object stage control module share the multi-channel spectrum gray level image acquisition module and the high-precision three-axis object stage;
the multichannel spectrum gray level image acquisition module has the following channels, wherein the number of the channels is N, and N is more than or equal to 2: the device comprises a long-wave-pass dichroic mirror, a narrow-band filter, a tube mirror and an image sensor.
And the channels of the multi-channel spectrum gray level image acquisition module are in conjugate positions with each other.
The multiple channels of the multi-channel spectrum gray level image acquisition module can simultaneously acquire N (N is more than or equal to 2) sample gray level images I with different central wavelength spectrum wave bands at zero time differencen(X, Y), X is more than or equal to 0 and less than or equal to X, Y is more than or equal to 0 and less than or equal to Y, N is more than or equal to 1 and less than or equal to N, wherein X is the total row number of the spectrum gray image, and Y is the total column number of the spectrum gray image.
The longitudinal dispersion enhanced optical imaging module comprises at least one objective lens with longitudinal dispersion, or comprises at least one tube lens with longitudinal dispersion, or other beneficial combinations.
The longitudinal dispersion enhanced optical imaging module has different focal lengths or image distances for optical signals of different wave bands, namely, under the same object distance, the object clear imaging axial positions of different wave bands are different, and the object surface height can be reversely deduced according to the clear imaging wave bands of the multispectral image sensor.
The longitudinal dispersion enhanced optical imaging module needs to eliminate transverse dispersion.
The high-precision three-axis objective table can realize high-precision longitudinal Z-axis motion and horizontal X-axis and Y-axis motion.
The large-field microscopic 3D appearance multichannel measuring method realized on the large-field microscopic 3D appearance multichannel measuring device comprises the following steps:
and 11, after the surface appearance of the sample in one observation field is restored, controlling the high-precision three-axis objective table to move along the X axis and the Y axis in the horizontal direction through the image analysis and objective table control system, switching to the next observation field, and repeating the operations from the step 8 to the step 10. If the sample surface appearance reduction of all observation fields is completed, the operation of the step 12 is carried out;
and step 12, carrying out image splicing on the microscopic 3D surface appearances under all observation fields to finish the large-field microscopic 3D appearance multi-channel measurement.
The large-field microscopic 3D appearance multichannel measuring method further comprises a method for judging the defocusing direction of the sample in the automatic focusing process in the use of the large-field microscopic 3D appearance multichannel measuring device, and specifically comprises the following steps:
(1) when a sample is focused, the spectral band with the maximum image definition value is taken as a central band;
(2) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is greater than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is smaller;
(3) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is smaller than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is larger;
(4) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is greater than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is smaller;
(5) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is smaller than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is larger;
(6) if the two adjacent spectral bands with the maximum definition do not comprise the central band and the two bands are smaller than the central band, the point is on a positive defocusing surface of the optical imaging system, and the defocusing amount is large;
(7) if the two adjacent spectral bands with the highest definition do not include the central band and the two bands are larger than the central band, the point is on the negative defocusing surface of the optical imaging system and the defocusing amount is large.
The large-field microscopic 3D morphology multi-channel measuring method further comprises a multi-channel spectral image gray difference ID(x, y) and a longitudinal height ZnThe relation curve calibration method comprises the following operation steps:
step 10.1, adjusting the high-precision three-axis objective table, and simultaneously obtaining axial characteristic curves I of axial light intensity and defocusing amount of central wavelength spectrum wave bands of different spectrum image acquisition channelsλn，1≤n≤N；
Step 10.2, acquiring axial characteristic curve I of axial light intensity and defocusing amount of central wavelength spectrum wave band of different spectrum image acquisition channelsλnCarrying out normalization processing;
step 10.3, in wave band lambda1，λ2For example, the actual operation is not limited to λ1，λ2Performing difference processing I on gray level images of different center wavelength spectrum wave bands lambda 1 and lambda 2λ1-Iλ2Obtaining the wave band lambda1，λ2Differential curve ID；
Step 10.4, for differential curve IDLinear function fitting is carried out in the linear region to obtain the gray difference I of the multi-channel spectral imageDAnd a longitudinal height ZnCalibration curve of the relationship.
The large-field microscopic 3D morphology multi-channel measuring method is characterized by comprising the following steps: the method can also comprise the step of correcting the illumination unevenness of the optical imaging system, wherein the correction processing mode is mainly realized by dividing the obtained gray difference or differential curve by the sum of the gray values of the corresponding points of the two-waveband gray image.
The large-field microscopic 3D morphology multi-channel measuring method is characterized by comprising the following steps: and correcting the uneven reflectivity of the samples, wherein the correcting method is mainly realized by multiplying the gray value of the gray image of the spectral band with different central wavelengths obtained in the step by a relative reflection coefficient. The relative reflection coefficient is set to 1 based on the substance with the largest surface reflectivity, and the relative reflection coefficient of other substances is the substance highest reflectivity divided by the substance reflectivity. The gray value of the gray image of the substances with the same height and different reflectivity under the same wave band can be equal through the correction processing mode.
The large-field microscopic 3D morphology multi-channel measuring method is characterized by comprising the following steps: the method can also comprise the step of compensating the gray level of the images of different spectral bands of the multi-channel spectral imaging system due to the fact that the transmittance of a filter changes with the band, the intensity of illumination light changes with the band, or the quantum effect of a multi-spectral camera changes with the band, wherein the compensation processing mode is mainly realized by multiplying the gray level of the gray level images of different central wavelength spectral bands obtained in the step by a band adjusting coefficient. The band adjustment coefficient obtaining mode is as follows: under the illumination of a uniform multi-color light source in a specific given space, a multi-spectral camera is adopted to obtain N spectral images from a surface-flattened sample with uniform reflectivity for different wave bands, the gray scale adjustment coefficient of the maximum gray scale image is 1, and the gray scale adjustment coefficients of other N-1 wave band images are the gray scale mean value of the maximum gray scale image divided by the gray scale mean value of the wave band image.
Compared with the existing microscopic 3D topography measurement technology, the invention adopts non-contact scanning, non-single-point scanning or layer-by-layer scanning, and can realize the large-view-field microscopic 3D topography measurement of the sample without axial movement of the object carrying table after primary automatic focusing. The method can realize microscopic 3D shape measurement with high transverse resolution, high longitudinal measurement precision, millimeter-scale longitudinal measurement range and inch-scale large-field transverse measurement range.
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FIG. 1 is a schematic structural diagram of a large-field microscopic 3D topography multi-channel measuring device.
FIG. 2 is a schematic structural diagram of a large-field microscopic 3D topography three-channel measuring device.
In the figure: the system comprises a 1-polychromatic light source, a 2-condenser, a 3-uniform collimating light lens group, a 4-semi-reflecting semi-permeable spectroscope, a 5-objective lens with longitudinal dispersion, a 6-high-precision three-axis objective table, a 7-tube lens with longitudinal dispersion, an 8-long-wave-pass dichroic mirror I, a 9-narrow-band filter I, a 10-tube lens I, an 11-image sensor I, a 12-long-wave-pass dichroic mirror II, a 13-narrow-band filter II, a 14-tube lens II, a 15-image sensor II, a 16-narrow-band filter N or narrow-band filter III, a 17-tube lens N, an 18-image sensor N and a 19-image analysis and objective table control system.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The structural schematic diagram of the large-field microscopic 3D morphology multichannel measuring device is shown in the attached figure 1 of the specification, and the device mainly comprises a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module and an image analysis and objective table control module. The longitudinal dispersion enhanced optical imaging module includes a multi-channel spectrum grayscale image acquisition module, and a plurality of spectrum grayscale image acquisition channels of the multi-channel spectrum grayscale image acquisition module may be provided, which will be further described below with reference to specific embodiments.
Detailed description of the preferred embodiment
The present embodiment is an apparatus embodiment.
The embodiment specifically sets three spectral grayscale image acquisition channels to obtain a large-field microscopic 3D morphology three-channel measurement device, and the structural schematic diagram is shown in fig. 2 in the specification.
The compound color light illumination module is sequentially provided with the following components in the light path propagation direction: the device comprises a polychromatic light source 1, a condenser 2, a uniform collimating light lens group 3, a semi-reflecting and semi-transmitting spectroscope 4 and an objective lens 5 with longitudinal dispersion.
The longitudinal dispersion enhanced optical imaging module is sequentially provided with the following components in the light path propagation direction: the device comprises a high-precision three-axis objective table 6, an objective lens 5 with longitudinal dispersion, a semi-reflecting and semi-transmitting spectroscope 4, a tube lens 7 with longitudinal dispersion and a multi-channel spectrum gray image acquisition module.
The image analysis and objective table control module is sequentially provided with the following components in a signal transmission direction: the system comprises a multi-channel spectrum gray level image acquisition module, an image analysis and object stage control system 19 and a high-precision three-axis object stage 6.
The polychromatic light illumination module and the longitudinal dispersion enhanced optical imaging module share a semi-reflecting semi-transparent spectroscope 4 and an objective lens 5 with longitudinal dispersion.
The longitudinal dispersion enhanced optical imaging module and the image analysis and object stage control module share the multi-channel spectrum gray level image acquisition module and the high-precision three-axis object stage 6.
The longitudinal dispersion enhanced optical imaging module comprises at least one objective lens 5 with longitudinal dispersion, or comprises at least one tube mirror 7 with longitudinal dispersion, or other beneficial combinations.
The longitudinal dispersion enhanced optical imaging module has different focal lengths or image distances for optical signals of different wave bands, namely, under the same object distance, the object clear imaging axial positions of different wave bands are different, and the object surface height can be reversely deduced according to the clear imaging wave bands of the multispectral image sensor.
The longitudinal dispersion enhanced optical imaging module needs to eliminate transverse dispersion.
The high-precision three-axis objective table 6 can realize high-precision longitudinal Z-axis motion and horizontal X-axis and Y-axis motion.
The multichannel spectrum gray image acquisition module of the specific embodiment of the large-field microscopic 3D morphology three-channel measurement device has 3 channels, and comprises: the device comprises a long-wave-pass dichroic mirror, a narrow-band filter, a tube mirror and an image sensor.
The 3 groups of spectral gray level image acquisition channels are respectively marked as I, II and III, wherein,
spectrum grey level image acquisition passageway I according to light path propagation direction, includes: the system comprises a dichroic mirror I8, a narrow-band filter I9, a tube mirror I10 and an image sensor I11;
spectrum grey level image acquisition channel II according to light path propagation direction, includes: a dichroic mirror II 12, a narrow-band filter II 13, a tube mirror II 14 and an image sensor II 15;
spectrum grey level image acquisition passageway III according to light path propagation direction, includes: a dichroic mirror II 12, a narrow-band filter III 16, a tube mirror III 17 and an image sensor III 18.
And the spectrum gray level image acquisition channel II and the spectrum gray level image acquisition channel III share a dichroic mirror II 12.
The 3 groups of spectral gray image acquisition channels are in conjugate positions with each other.
The 3 groups of spectral gray image acquisition channels can acquire the spectral gray image I of the sample under 3 different spectral wave bands with zero time differencen(X, Y), X is more than or equal to 0 and less than or equal to X, Y is more than or equal to 0 and less than or equal to Y, and n is 1,2 and 3, wherein X is the total number of rows of the spectrum band gray-scale image, and Y is the total number of columns of the spectrum band gray-scale image.
Detailed description of the invention
This embodiment is an embodiment of a method implemented on the apparatus described in the first embodiment.
The three-channel measurement method for the large-field microscopic 3D topography comprises the following steps:
and 11, after the surface appearance of the sample in one observation field is restored, controlling the high-precision three-axis objective table 6 to move along the X axis and the Y axis in the horizontal direction through the image analysis and objective table control system 19, switching to the next observation field, and repeating the operations from the step 8 to the step 10. If the sample surface appearance reduction of all observation fields is completed, the operation of the step 12 is carried out;
and step 12, carrying out image splicing on the microscopic 3D surface appearances under all observation fields to finish the large-field microscopic 3D appearance multi-channel measurement.
The three-channel measurement method for the large-field microscopic 3D topography of the embodiment further includes a method for determining the defocusing direction of a sample in an automatic focusing process in use of the three-channel measurement device for the large-field microscopic 3D topography, which specifically includes:
(1) when a sample is focused, the spectral band with the maximum image definition value is taken as a central band;
(2) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is greater than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is smaller;
(3) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is smaller than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is larger;
(4) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is greater than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is smaller;
(5) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is smaller than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is larger;
(6) if the two adjacent spectral bands with the maximum definition do not comprise the central band and the two bands are smaller than the central band, the point is on a positive defocusing surface of the optical imaging system, and the defocusing amount is large;
(7) if the two adjacent spectral bands with the highest definition do not include the central band and the two bands are larger than the central band, the point is on the negative defocusing surface of the optical imaging system and the defocusing amount is large.
The large-field microscopic 3D morphology multi-channel measurement method of the embodiment also comprises a multi-channel spectral image gray difference ID(x, y) and a longitudinal height ZnThe relation curve calibration method comprises the following steps:
step 10.1, adjusting the high-precision three-axis objective table 6, and simultaneously obtaining axial characteristic curves I of axial light intensity and defocusing amount of central wavelength spectrum wave bands of 3 groups of spectrum image acquisition channelsλn，1≤n≤N；
Step 10.2, carrying out axial characteristic curve I of axial light intensity and defocusing amount of central wavelength spectrum wave band of 3 groups of spectrum image acquisition channelsλnCarrying out normalization processing;
step 10.3, in wave band lambda1，λ2For example, the actual operation is not limited to λ1，λ2Performing difference processing I on gray level images of different center wavelength spectrum wave bands lambda 1 and lambda 2λ1-Iλ2Obtaining the wave band lambda1，λ2Differential curve ID；
Step 10.4, for differential curve IDLinear function fitting is carried out in the linear region to obtain the gray difference I of the multi-channel spectral imageDAnd a longitudinal height ZnCalibration curve of the relationship.
The three-channel measurement method for the large-field microscopic 3D topography of the embodiment further includes correction processing of the unevenness of illumination light of the optical imaging system, wherein the correction processing mode is mainly realized by dividing the obtained gray difference or differential curve by the sum of gray values of corresponding points of the two-band gray image.
The three-channel measurement method for the large-field microscopic 3D morphology further comprises the step of correcting the uneven reflectivity of the samples, wherein the correction processing mode is mainly realized by multiplying the gray values of the gray images of the spectral bands with different central wavelengths obtained in the step by a relative reflection coefficient. The relative reflection coefficient is set to 1 based on the substance with the largest surface reflectivity, and the relative reflection coefficient of other substances is the substance highest reflectivity divided by the substance reflectivity. The gray value of the gray image of the substances with the same height and different reflectivity under the same wave band can be equal through the correction processing mode.
The three-channel measurement method for the large-field microscopic 3D morphology further comprises the step of compensating the gray scale of images of different spectral bands of the multi-channel spectral imaging system due to the fact that the transmittance of a filter changes with the band, or the intensity of illumination light changes with the band, or the quantum effect of a multi-spectral camera changes with the band, wherein the compensation processing mode is mainly realized by multiplying the gray scale value of the gray scale image of the spectral band of different central wavelengths obtained in the step by a band adjustment coefficient. The band adjustment coefficient obtaining mode is as follows: under the illumination of a uniform multi-color light source in a specific given space, a multi-spectral camera is adopted to obtain N spectral images from a surface-flattened sample with uniform reflectivity for different wave bands, the gray scale adjustment coefficient of the maximum gray scale image is 1, and the gray scale adjustment coefficients of other N-1 wave band images are the gray scale mean value of the maximum gray scale image divided by the gray scale mean value of the wave band image.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A large-field microscopic 3D appearance multichannel measuring device is characterized in that: comprises a polychromatic light illumination module, a longitudinal dispersion enhanced optical imaging module and an image analysis and objective table control module,
the compound color light illumination module is sequentially provided with the following components in the light path propagation direction: the device comprises a polychromatic light source (1), a condenser (2), a uniform collimating light lens group (3), a semi-reflecting and semi-transmitting spectroscope (4) and an objective lens (5) with longitudinal dispersion;
the longitudinal dispersion enhanced optical imaging module is sequentially provided with the following components in the light path propagation direction: the device comprises a high-precision three-axis objective table (6), an objective lens (5) with longitudinal dispersion, a semi-reflecting and semi-transmitting spectroscope (4), a tube lens (7) with longitudinal dispersion and a multi-channel spectrum gray image acquisition module;
the image analysis and objective table control module is sequentially provided with the following components in a signal transmission direction: the system comprises a multi-channel spectrum gray level image acquisition module, an image analysis and objective table control system (19) and a high-precision three-axis objective table (6);
the polychromatic light illumination module and the longitudinal dispersion enhanced optical imaging module share the semi-reflecting semi-transparent spectroscope (4) and the objective lens (5) with longitudinal dispersion;
the longitudinal dispersion enhanced optical imaging module and the image analysis and object stage control module share the multi-channel spectrum gray level image acquisition module and the high-precision three-axis object stage (6);
the multichannel spectrum gray level image acquisition module has N channels, wherein N is more than or equal to 2, and each channel comprises: the device comprises a long-wave-pass dichroic mirror, a narrow-band filter, a tube mirror and an image sensor.
2. The large-field-of-view microscopic 3D topography multi-channel measurement device according to claim 1, wherein: and the channels of the multi-channel spectrum gray level image acquisition module are in conjugate positions with each other.
3. The large-field-of-view microscopic 3D topography multi-channel measurement device according to claim 1, wherein: multiple channels of the multi-channel spectrum gray level image acquisition module can simultaneously acquire N sample gray level images I with different central wavelength spectrum wave bands at zero time differencen(X, Y), wherein N is more than or equal to 2, X is more than or equal to 0 and less than or equal to X, Y is more than or equal to 0 and less than or equal to Y, N is more than or equal to 1 and less than or equal to N, X is the total row number of the spectrum gray image, and Y is the total column number of the spectrum gray image.
4. The large-field-of-view microscopic 3D topography multi-channel measurement device according to claim 1, wherein: the longitudinal dispersion enhanced optical imaging module comprises at least one objective lens (5) with longitudinal dispersion or at least one tube lens (7) with longitudinal dispersion.
5. The large-field-of-view microscopic 3D topography multi-channel measurement device according to claim 1, wherein: the longitudinal dispersion enhanced optical imaging module needs to eliminate transverse dispersion.
6. The large-field-of-view microscopic 3D topography multi-channel measurement device according to claim 1, wherein: the high-precision three-axis objective table (6) can realize high-precision longitudinal Z-axis motion and horizontal X-axis and Y-axis motion.
7. A large-field-of-view microscopic 3D topography multi-channel measurement method, characterized in that the large-field-of-view microscopic 3D topography multi-channel measurement device of any one of claims 1 to 6 is used, comprising the steps of:
step 1, a sample to be detected is placed on a high-precision three-axis objective table (6);
step 2, adjusting the high-precision three-axis objective table (6) to enable a certain spectral band imaging channel of the multi-channel spectral gray image acquisition module to clearly image the sample;
step 3, acquiring N samples in spectral band gray level images I with different central wavelengths by the multi-channel spectral gray level image acquisition modulen(X, Y), wherein N is more than or equal to 2, X is more than or equal to 0 and less than or equal to X, Y is more than or equal to 0 and less than or equal to Y, N is more than or equal to 1 and less than or equal to N, X is the total row number of the spectrum band gray level images, and Y is the total column number of the spectrum band gray level images;
step 4, calculating the definition value F of each spectrum gray level imagenThe sharpness value FnThe method can be used for calculating according to any one of the image definition evaluation functions of the Laplacian function, the Brenner function and the Tenengrad function;
step 5, collecting the spectrum gray image definition values F of two adjacent channels with the maximum definitionnPerforming difference processing to obtain a defocus differential signal FD＝Fn-1-Fn；
Step 6, through the out-of-focus differential signal F of the scale in advanceDAcquiring a defocusing amount value and a defocusing direction signal from a defocusing amount relation curve;
step 7, the image analysis and objective table control system (19) adjusts the high-precision three-axis objective table (6) to the focus surface of the optical imaging system according to the defocusing amount value and the defocusing direction signal to complete automatic focusing;
step 8, repeating the operation of the step 3;
step 9, performing difference processing on each point of the spectrum gray level images collected by the adjacent channels to obtain the gray level difference I of the multi-channel spectrum imagesD(x,y)＝In(x,y)-In-1(x,y)；
Step (ii) of10, through pre-calibrated multi-channel spectral image gray difference IDAnd a longitudinal height ZnCalculating a relation curve, and reducing the surface topography Z (x, y) of the sample;
step 11, after the reduction of the surface appearance of the sample in one observation field is finished, controlling a high-precision three-axis objective table (6) to move along the X axis and the Y axis in the horizontal direction through an image analysis and objective table control system (19), switching to the next observation field, and repeating the operation from the step 8 to the step 10; if the sample surface appearance reduction of all observation fields is completed, the operation of the step 12 is carried out;
and step 12, carrying out image splicing on the microscopic 3D surface appearances under all observation fields to finish the large-field microscopic 3D appearance multi-channel measurement.
8. The large-field-of-view microscopic 3D topography multi-channel measurement method according to claim 7, wherein: the method also comprises a method for judging the defocusing direction of the sample in the automatic focusing process in the use of the large-field microscopic 3D morphology multi-channel measuring device, and the specific judging process is as follows:
(1) when a sample is focused, the spectral band with the maximum image definition value is taken as a central band;
(2) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is greater than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is smaller;
(3) if one of the two adjacent spectral bands with the maximum definition is a central band which is a longer band, and the definition value of the spectral image of the central band is smaller than that of the spectral image of the other band, the point is on a positive defocusing surface of the optical imaging system and the defocusing amount is larger;
(4) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is greater than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is smaller;
(5) if one of the two adjacent spectral bands with the maximum definition is a central band which is a shorter band, and the spectral image definition value of the central band is smaller than that of the other band, the point is on a negative defocusing surface of the optical imaging system and the defocusing amount is larger;
(6) if the two adjacent spectral bands with the maximum definition do not comprise the central band and the two bands are smaller than the central band, the point is on a positive defocusing surface of the optical imaging system, and the defocusing amount is large;
(7) if the two adjacent spectral bands with the highest definition do not include the central band and the two bands are larger than the central band, the point is on the negative defocusing surface of the optical imaging system and the defocusing amount is large.
9. The large-field-of-view microscopic 3D topography multi-channel measurement method according to claim 7, wherein: the method can further comprise the step of multi-channel spectral image gray difference ID(x, y) and a longitudinal height ZnThe relation curve calibration method comprises the following operation steps:
step 10.1, adjusting the high-precision three-axis objective table (6), and simultaneously obtaining axial characteristic curves I of axial light intensity and defocusing amount of central wavelength spectrum wave bands of different spectrum image acquisition channelsλn，1≤n≤N；
Step 10.2, acquiring axial characteristic curve I of axial light intensity and defocusing amount of central wavelength spectrum wave band of different spectrum image acquisition channelsλnCarrying out normalization processing;
step 10.3, spectral bands lambda of different central wavelengths1，λ2The gray level image is subjected to difference processing Iλ1-Iλ2Obtaining the wave band lambda1，λ2Differential curve ID；
Step 10.4, for differential curve IDLinear function fitting is carried out in the linear region to obtain the gray difference I of the multi-channel spectral imageDAnd a longitudinal height ZnCalibration curve of the relationship.
10. The large-field-of-view microscopic 3D topography multi-channel measurement method according to any one of claims 7 to 9, wherein: the method also comprises the step of correcting the illumination unevenness of the optical imaging system, wherein the correction processing mode is realized by dividing the obtained gray difference or differential curve by the sum of the gray values of corresponding points of the gray images of the two wave bands, the correction processing mode for resisting the sample reflectivity unevenness is realized by multiplying the gray values of the obtained gray images of the different central wavelength spectrum wave bands by a relative reflection coefficient, the compensation processing mode is also realized by multiplying the gray values of the different spectrum wave band images of the multi-channel spectrum imaging system by a wave band adjusting coefficient, and the compensation processing mode is realized by multiplying the gray values of the obtained gray images of the different central wavelength spectrum wave bands by the wave band adjusting coefficient.
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