CN111999878B - Microscopic imaging system and real-time focusing method thereof - Google Patents
Microscopic imaging system and real-time focusing method thereof Download PDFInfo
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Abstract
The invention relates to a microscopic imaging system and a real-time focusing method thereof, wherein the microscopic imaging system comprises: a primary light source; the collecting lens is positioned above the main light source, and the optical axis of the collecting lens and the optical axis of the main light source are overlapped; the imaging unit is positioned above the collecting lens, and the optical axis of the imaging unit and the optical axis of the collecting lens are overlapped; further comprising: an auxiliary light source for generating a diffraction ring in an image acquired by the imaging unit; the auxiliary light source is located on a focal plane below the condenser lens, and the auxiliary light source deviates from an optical axis of the condenser lens. By adopting the invention, the focusing precision and efficiency of the microscopic imaging system are ensured.
Description
The application has application date of 2017, 11 and 16, application number of 201711133587.3, and invention name: a microscopic imaging system and a divisional application of a real-time focusing method thereof are provided.
Technical Field
The present invention relates to an imaging system and a focusing method thereof, and more particularly, to a microscopic imaging system and a focusing method thereof.
Background
In the microscopic imaging process, the objective lens is required to be kept all the time to be capable of clearly imaging the sample. Therefore, focusing in a microscopic imaging system is a prerequisite. In the prior art, the automatic focusing technology is mainly divided into two categories: one is a passive focusing method based on image gray contrast analysis. Another type is an active focusing method based on a ranging method. The passive focusing method is realized by obtaining a plurality of images by changing the distance between the camera and the target object and analyzing the characteristics of the images. The method needs to make the moving distance of the lens long, continuously collect a plurality of images to calculate the definition of corresponding positions to form a curve, and judge the focus position according to the maximum value of the curve. The passive focusing method has a large calculation amount, requires multiple lens displacements, and is difficult to meet the requirement of rapidness and accuracy in the real-time focusing process. The active focusing method realizes focusing by adding an additional signal transmitting and receiving device, and infrared ranging, ultrasonic ranging, triangular ranging and the like are common. When the detected target has strong absorption effect on infrared light or ultrasonic waves, the active focusing method is easy to malfunction or inaccurate in focusing. Especially for micro-focusing, the use of high-magnification objective lens greatly reduces the focal depth (in micron order), and the precision of infrared distance measurement, ultrasonic distance measurement and triangular distance measurement is difficult to meet the requirement.
Disclosure of Invention
The invention aims to provide a microscopic imaging system and a focusing method thereof, and solves the problem of poor focusing precision of the microscopic imaging system.
To achieve the above object, the present invention provides a microscopic imaging system comprising:
a primary light source;
the collecting lens is positioned above the main light source, and the optical axis of the collecting lens and the optical axis of the main light source are overlapped;
the imaging unit is positioned above the collecting lens, and the optical axis of the imaging unit and the optical axis of the collecting lens are overlapped;
further comprising:
an auxiliary light source for generating a diffraction ring in an image acquired by the imaging unit;
the auxiliary light source is located on a focal plane below the condenser lens, and the auxiliary light source deviates from an optical axis of the condenser lens.
According to an aspect of the present invention, the imaging unit includes:
the objective lens is positioned above the condenser lens, and the optical axis of the objective lens and the optical axis of the condenser lens are overlapped;
and the polymerization lens is positioned above the objective lens, and the optical axis of the polymerization lens and the optical axis of the objective lens are superposed with each other.
According to an aspect of the invention, further comprising:
an object stage positioned between the objective lens and the condenser lens;
the imaging camera is positioned above the polymerization mirror, and the optical axis of the imaging camera and the optical axis of the polymerization mirror are superposed;
a central control unit connected with the imaging unit;
and the driving device is used for adjusting the distance between the objective lens and the objective table.
In order to achieve the above object, the present invention provides a real-time focusing method, comprising:
s1, enabling an objective table to be located at a focal position of an objective lens;
s2, adjusting the distance between the objective lens and the objective table at a preset interval distance, and acquiring a sample defocusing image through the objective lens;
s3, generating an out-of-focus autocorrelation image with a diffraction ring according to the out-of-focus image of the sample, and acquiring a radius value of the diffraction ring;
s4, repeating S2-S3, and obtaining a defocusing relation curve according to the radius value and the preset interval distance;
s5, obtaining a sample image, and obtaining a radius value of a diffraction ring of the sample image according to the sample image;
s6, controlling the objective table (5) to be kept at the focal position of the objective lens (31) according to the defocusing relation curve.
According to an aspect of the present invention, the step of S1 includes:
s11, opening a main light source to enable the objective table to be located at the focal position of the objective lens;
and S12, closing the main light source, opening the auxiliary light source, and acquiring an in-focus image of the sample on the objective table through an imaging camera.
According to an aspect of the present invention, the step of S3 includes:
s31, carrying out Fourier transform on the sample out-of-focus image to obtain power spectrum information of the sample out-of-focus image;
s32, carrying out inverse Fourier transform on the power spectrum information of the sample defocused image to obtain an intermediate image;
s33, performing autocorrelation processing on the intermediate image to generate a defocused light intensity distribution map;
and S34, acquiring the radius value of the diffraction ring according to the pixel difference between the light intensity major maximum and the light intensity minor maximum in the defocusing light intensity distribution graph.
According to an aspect of the present invention, by using the diffraction light generation unit of the present invention, the micro-imaging system can obtain the radius of the diffraction ring by performing autocorrelation processing on the defocused image in the defocused state. And generating a light intensity distribution graph according to the defocused autocorrelation image after autocorrelation processing, and calculating the radius value of the first-order diffraction ring according to the pixel value between the light intensity main maximum and the light intensity secondary maximum in the light intensity distribution graph. The radius value of the first-order diffraction ring obtained according to the pixel value between the light intensity main maximum and the light intensity secondary maximum is quick and accurate, the real-time focusing of the microscopic imaging system is realized, and the focusing precision and efficiency of the microscopic imaging system are ensured.
According to one scheme of the invention, the diffraction light generating unit has a simple structure and is convenient to assemble and disassemble. Through the arrangement mode of the diffraction light generating unit, when the diffraction light generating unit is arranged in a microscopic imaging system, the change of the microscopic imaging system is small, so that the imaging stability of the limiting imaging system and the structural stability of the microscopic imaging system are ensured. Meanwhile, the diffraction light generating unit saves cost and is easy to realize.
Drawings
FIG. 1 schematically shows a block diagram of a microscopy imaging system according to an embodiment of the invention;
FIG. 2 is a block diagram schematically illustrating steps of a real-time focusing method according to an embodiment of the present invention;
FIG. 3 schematically shows an in-focus autocorrelation of a sample of a microscopy imaging system according to one embodiment of the present invention;
FIG. 4 schematically shows an in-focus intensity profile of a sample of a microscopy imaging system according to an embodiment of the invention;
FIG. 5 is a schematic diagram illustrating the distribution of the position of different diffraction light generating units and the intensity of sample at focus in a microscopic imaging system according to an embodiment of the present invention;
FIG. 6 is a block diagram schematically illustrating an out-of-focus image processing step of a real-time focusing method according to an embodiment of the present invention;
FIG. 7 schematically shows a sample through focus autocorrelation of a microscopic imaging system according to one embodiment of the present invention;
FIG. 8 schematically shows a sample through focus intensity profile of a microscopic imaging system according to an embodiment of the present invention;
FIG. 9 schematically shows a sample through-focus light intensity distribution plot at different through-focus amounts for a microscopic imaging system according to an embodiment of the present invention;
FIG. 10 schematically shows a graph of defocus curves for a microscopy imaging system according to an embodiment of the invention.
Detailed Description
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
In describing embodiments of the present invention, the terms "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer" are used in an orientation or positional relationship that is illustrated in the associated drawings for convenience and simplicity of description only, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus are not to be considered limiting of the present invention.
The present invention is described in detail below with reference to the drawings and the specific embodiments, which are not repeated herein, but the embodiments of the present invention are not limited to the following embodiments.
As shown in fig. 1, according to one embodiment of the present invention, the microscopic imaging system of the present invention includes a main light source 1, a condenser lens 2, an imaging unit 3, a diffracted light generating unit 4, a stage 5, an imaging camera 6, a central control unit 7, and a driving device 8. In the present embodiment, the optical axis of the condenser lens 2 and the optical axis of the main light source 1 overlap each other, and the condenser lens 2 is positioned above the main light source 1. The optical axis of the imaging unit 3 coincides with the optical axis of the condenser lens 2, and the imaging unit 3 is located above the condenser lens 2. The stage 5 is located between the imaging unit 3 and the condenser lens 2. The optical axis of the imaging camera 6 coincides with the optical axis of the imaging unit 3, and the imaging camera 6 is located above the imaging unit 3. The central control unit 7 is connected with the imaging camera 6 and the driving device 8, respectively. The image taken by the imaging camera 6 can be acquired by the central control unit 7, and the distance between the stage 5 and the imaging unit 3 is adjusted by the central control unit 7 controlling the driving device 8, enabling the imaging unit 3 to perform focusing.
As shown in fig. 1, according to one embodiment of the present invention, the imaging unit 3 includes an objective lens 31 and a condenser lens 32. In the present embodiment, the optical axis of the objective lens 31 coincides with the optical axis of the condenser lens 2, and the objective lens 31 is positioned above the condenser lens 2. The optical axis of the condenser lens 32 coincides with the optical axis of the objective lens 31, and the condenser lens 32 is located above the objective lens 31. In the present embodiment, the sample a on the stage 5 is imaged by the objective lens 31, and the resultant image is transmitted to the imaging camera 6 by the converging lens 32.
As shown in fig. 1, according to one embodiment of the present invention, the diffracted light generating unit 4 is located below the condenser lens 2. In the present embodiment, the diffracted light generation unit 4 includes the auxiliary light source 41. The auxiliary light source 41 is located on the focal plane below the condenser lens 2, and the auxiliary light source 41 is offset from the optical axis of the condenser lens 2.
To further explain the present invention in detail, the focusing method of the imaging system of the present invention is explained in detail with reference to the accompanying drawings.
As shown in fig. 2, the real-time focusing method according to the present invention includes:
s1, positioning an objective table 5 at the focal position of an objective lens 31;
s2, adjusting the distance between the objective lens 31 and the objective table 5 at a preset interval distance to obtain a defocused image;
s3, generating an out-of-focus autocorrelation image with a diffraction ring according to the out-of-focus image, and acquiring a radius value of the diffraction ring;
s4, repeating S2-S3, and obtaining a defocusing relation curve according to the radius value and the preset interval distance;
s5, acquiring a sample image according to the objective lens 31, and acquiring the radius value of the diffraction ring of the sample image
S6, controlling the objective table 5 to keep at the focal position of the objective lens 31 according to the defocusing relation curve. Referring to fig. 1 and 2, according to an embodiment of the present invention, the step S1 includes:
s11, the main light source 1 is turned on, and the objective table 5 is positioned at the focal position of the objective lens 31. In the present embodiment, the main light source 1 is turned on to image the sample a on the stage 5, and the central control unit 7 acquires an image of the sample a by the imaging camera 6. The central control unit 7 controls the driving device 8 to move according to the preset MTF curve, adjusts the distance between the object stage 5 and the object lens 31, and makes the object stage 5 located at the focal position of the object lens 31, so that the imaging camera 6 can obtain the sharpest image of the sample a. In the present embodiment, the driving device 8 may drive the stage 5 or the objective lens 31 to move in the vertical direction, so that the stage 5 is located at the focal position of the objective lens 31.
S12, turning off the main light source 1, turning on the auxiliary light source 41, and acquiring an in-focus image of the sample A on the objective table 5 through the imaging camera 6. In the present embodiment, the main light source 1 is turned off, the auxiliary light source 41 located at the focal plane position below the condenser lens 2 is turned on, and the auxiliary light source 41 emits oblique light. The central control unit 7 acquires an in-focus image of the sample a by the imaging camera 6 and acquires an in-focus autocorrelation image (i.e., a bright spot position in fig. 3) after the autocorrelation processing, and an in-focus light intensity distribution map (see fig. 4) by the in-focus autocorrelation image. As shown in fig. 4, when the auxiliary light source 41 emits oblique light, the sample acquired by the central control unit 7 has a step C on both sides of the intensity principal maximum B in the focal intensity distribution diagram of the focal image. By changing the position of the auxiliary light source 41 on the focal plane below the condenser lens 2 (except for the vertical intersection point of the optical axis of the condenser lens 2 and the focal plane), the value of the light intensity dominant pole B and the step-like step C on both sides of the light intensity dominant pole B are also changed. As shown in fig. 5, the sub-pictures in the figure sequentially show from (a) to (d), the value of the principal intensity B of the auxiliary light source 41 decreases and the width of the step C increases as the position on the focal plane below the condenser lens 2 is farther from the optical axis of the condenser lens 2.
In the present embodiment, the in-focus image is fourier-transformed before the autocorrelation processing is performed on the in-focus image, and the power spectrum information of the in-focus image is acquired. And performing inverse Fourier transform according to the acquired power spectrum information. After the focal image is subjected to autocorrelation processing, the intensity in the image after the autocorrelation processing is found, thereby generating an in-focus intensity distribution map.
Referring to fig. 1 and 2, according to an embodiment of the present invention, the distance between the objective lens 31 and the stage 5 is adjusted by a predetermined interval distance in S2. In the present embodiment, the predetermined spacing distance is set to 1 μm. The position of the auxiliary light source 41 is fixed, and the central control unit 7 controls the driving device 8 to drive the stage 5 or the objective lens 31 to move 1 micron in the vertical direction. The central control unit 7 acquires a sample-through-focus image of the sample a by the imaging camera 6.
Referring to fig. 1, 2 and 6, according to an embodiment of the present invention, in step S3, an out-of-focus autocorrelation image having a diffraction ring is generated from the sample out-of-focus image. In the present embodiment, the present invention further includes:
s31, carrying out Fourier transform on the sample out-of-focus image to obtain power spectrum information of the sample out-of-focus image;
s32, carrying out inverse Fourier transform on the power spectrum information of the sample defocused image to obtain an intermediate image;
s33, performing autocorrelation processing on the intermediate image to generate a defocusing light intensity distribution map;
and S34, acquiring the radius value of the diffraction ring according to the pixel difference between the light intensity major maximum and the light intensity minor maximum in the defocused light intensity distribution diagram.
In the present embodiment, the intermediate image is subjected to autocorrelation processing to generate a defocus autocorrelation image (see fig. 7), and as can be seen from a comparison of fig. 3 and 7, a semicircular diffraction ring appears around the image of the sample a in the defocus autocorrelation image, and in the present embodiment, the semicircular diffraction ring is a first-order diffraction ring by the aforementioned diffraction light generation unit 4. A defocus intensity distribution map (see fig. 8) is generated from the defocus autocorrelation image, and as shown in fig. 8, intensity sub-maxima D appear on both sides of the intensity main maximum B. The radius value of the diffraction ring can be obtained by calculating the distance between the light intensity main maximum B and the light front secondary maximum D.
As shown in fig. 7, according to an embodiment of the present invention, by adjusting the position of the auxiliary light source 41, the center line of the half-ring chord of two diffracting rings in the off-focus autocorrelation image is aligned with the imaging center of the sample, so that the center line of the half-ring chord of the diffracting ring is parallel to the CCD image plane, i.e. the center line of the half-ring chord of the diffracting ring is parallel to the edge of the off-focus autocorrelation image, thereby facilitating the subsequent measurement of the radius of the diffracting ring and facilitating the calculation of the radius of the diffracting ring.
As shown in fig. 1, according to an embodiment of the present invention, by changing the position of the auxiliary light source 41, one or two semicircular diffraction rings appear around the image of the sample a in the out-of-focus autocorrelation image.
Referring to fig. 1 and 2, in step S4, repeating steps S2-S3, and obtaining a defocus relationship curve according to the radius value and the predetermined separation distance. In the present embodiment, step S2 is repeated, the central control unit 7 controls the driving device 8 to move the objective lens 31 or the stage 5 in the same direction a plurality of times (two times, three times or more) at intervals of a distance of 1 μm, and the central control unit 7 acquires sample-off-focus images of a plurality of (two, three or more) samples a by the imaging camera 6. The central control unit 7 acquires a sample-off-focus image of one sample a every time the driving device 8 moves. By adopting the embodiment in step S3, a plurality of (two, three, or more) defocus autocorrelation images are acquired, and a plurality of (two, three, or more) defocus intensity distribution maps are generated from the plurality of (two, three, or more) defocus autocorrelation images. As shown in fig. 9, the defocus intensity distribution diagram having four different defocus distances of the same kind is obtained by sequentially performing (a) to (D) from the subpicture, the distance between the objective lens 31 and the stage 5 is gradually increased by the driving device 8, the distance between the light intensity secondary maximum D and the light intensity main maximum B is gradually increased, and the value component of the light intensity main maximum B is decreased. In the present embodiment, the central control unit 7 needs to acquire sample off-focus images of 30 samples a by the imaging camera 6, that is, the total length of the driving device 8 that needs to travel in the same direction is 30 micrometers.
In the present embodiment, the acquired sample out-of-focus images of 30 samples a are all subjected to autocorrelation processing to generate out-of-focus autocorrelation images, and a light intensity distribution map is acquired. From the obtained 30 intensity profiles, the pixel difference values (i.e., the radius values of the diffraction ring) between the 30 intensity secondary maxima D and the intensity primary maxima B are obtained. As shown in fig. 10, a coordinate point E is plotted in a rectangular coordinate game according to the defocus amount (i.e., the length of travel of the driving device 8 at a predetermined interval distance) and the radius value (i.e., the pixel difference between the light intensity secondary maximum D and the light intensity primary maximum B), and the plotted coordinate point E is fitted to a curve to generate a defocus relationship curve F.
In the present embodiment, the defocus relationship curve F generated by fitting satisfies:
y＝kx+b
wherein y is defocus and x is the radius of the diffraction ring. After the radius of the diffraction ring is measured, the solved k and b values can be fitted by utilizing the defocusing relation curve. And obtaining the value of the radius x of the diffraction ring according to the formula, namely obtaining the corresponding value of the defocusing amount.
According to one embodiment of the present invention, in step S5, the stage 5 is held at the focal position of the objective lens 3 based on the curve F of the radius value and the defocus relationship. In the present embodiment, the central control unit 7 obtains the radius value (i.e. the distance between the light intensity secondary maximum D and the light intensity principal maximum B) of the diffraction ring in the processed image through the image of the sample a obtained by the imaging camera 6 and performing autocorrelation processing, and can obtain the defocus amount of the current microscopic imaging system according to the generated defocus relationship curve F. According to the acquired defocus amount, the central control unit 7 controls the driving device 8 to adjust the distance between the objective lens 31 and the stage 5 so that the stage 5 is always positioned at the focal point of the objective lens 31, thereby realizing real-time focusing of the imaging system.
According to the present invention, the diffraction light generating unit 4 has a simple structure and is easily assembled and disassembled. Through the arrangement mode of the diffraction light generating unit 4, when the diffraction light generating unit 4 is installed in a microscopic imaging system, the change of the microscopic imaging system is small, so that the imaging stability of the limiting imaging system and the structural stability of the microscopic imaging system are ensured. Meanwhile, the diffraction light generating unit 4 of the invention saves cost and is easy to realize.
According to the invention, by adopting the diffraction light generating unit 4, the radius of the diffraction ring can be obtained by performing autocorrelation processing on the defocused image in the defocused state of the microscopic imaging system, and the real-time focusing on the microscopic imaging system is realized by calculating the radius value of the diffraction ring, so that the focusing precision and efficiency of the microscopic imaging system are ensured.
The foregoing is illustrative of specific embodiments of the present invention and reference should be made to the implementation of apparatus and structures not specifically described herein, which is understood to be a general purpose apparatus and method of operation known in the art.
The above description is only one embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A microscopic imaging system, comprising:
a primary light source (1);
the condenser (2) is positioned above the main light source (1), and the optical axis of the condenser (2) and the optical axis of the main light source (1) are overlapped;
the imaging unit (3) is positioned above the collecting lens (2), and the optical axis of the imaging unit (3) and the optical axis of the collecting lens (2) are overlapped;
it is characterized by also comprising:
an auxiliary light source (41) for generating a diffraction ring in an image acquired by the imaging unit (3);
the auxiliary light source (41) is located on a focal plane below the condenser (2), and the auxiliary light source (41) deviates from an optical axis of the condenser (2).
2. Microscopic imaging system according to claim 1, characterized in that the imaging unit (3) comprises:
the objective lens (31) is positioned above the condenser lens (2), and the optical axis of the objective lens (31) and the optical axis of the condenser lens (2) are overlapped;
the converging lens (32) is positioned above the objective lens (31), and the optical axis of the converging lens (32) and the optical axis of the objective lens (31) are overlapped.
3. The microscopy imaging system of claim 2, further comprising:
an object stage (5), the object stage (5) being located between the objective lens (31) and the condenser lens (2);
an imaging camera (6), the imaging camera (6) is located above the converging mirror (32), and the optical axis of the imaging camera (6) and the optical axis of the converging mirror (32) are arranged in a mutual coincidence manner
A central control unit (7) connected with the imaging unit (3);
a drive device (8) for adjusting the distance between the objective lens (31) and the object stage (5).
4. A real-time focusing method using the microscopic imaging system of claim 3, comprising:
s1, enabling an objective table (5) to be located at a focal position of an objective lens (31);
s2, adjusting the distance between the objective lens (31) and the objective table (5) at a preset interval distance, and acquiring a sample defocused image through the objective lens (31);
s3, generating an out-of-focus autocorrelation image with a diffraction ring according to the out-of-focus image of the sample, and acquiring a radius value of the diffraction ring;
s4, repeating S2-S3, and obtaining a defocusing relation curve according to the radius value and the preset interval distance;
s5, obtaining a sample image, and obtaining a radius value of a diffraction ring of the sample image according to the sample image;
s6, controlling the objective table (5) to be kept at the focal position of the objective lens (31) according to the defocusing relation curve.
5. The real-time focusing method of the microscopic imaging system as claimed in claim 4, wherein the step of S1 includes:
s11, opening a main light source (1) to enable the objective table (5) to be located at the focal position of the objective lens (31);
s12, turning off the main light source (1), turning on the auxiliary light source (41), and acquiring an in-focus image of the sample on the objective table (5) through the imaging camera (6).
6. The real-time focusing method of the microscopic imaging system as claimed in claim 5, wherein the step of S3 includes:
s31, carrying out Fourier transform on the sample out-of-focus image to obtain power spectrum information of the sample out-of-focus image;
s32, carrying out inverse Fourier transform on the power spectrum information of the sample defocused image to obtain an intermediate image;
s33, performing autocorrelation processing on the intermediate image to generate a defocused light intensity distribution map;
and S34, acquiring the radius value of the diffraction ring according to the pixel difference between the light intensity major maximum and the light intensity minor maximum in the defocusing light intensity distribution graph.
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| CN111443477B (en) * | 2020-04-13 | 2022-12-20 | 腾讯科技（深圳）有限公司 | Microscope auto-focusing method, microscope system, medical device, and storage medium |
| CN116430568A (en) * | 2020-04-13 | 2023-07-14 | 腾讯科技（深圳）有限公司 | Microscope system, microscope auto-focusing method, and medical apparatus |
| CN112859317A (en) * | 2021-01-20 | 2021-05-28 | 宁波舜宇仪器有限公司 | Automatic focusing microscopic imaging system |
| CN112748564A (en) * | 2021-01-29 | 2021-05-04 | 上海睿钰生物科技有限公司 | Microscope device and focusing method thereof |
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| CN107656364B (en) | 2020-10-23 |
| CN111999878A (en) | 2020-11-27 |
| CN107656364A (en) | 2018-02-02 |
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