CN110716301B - Automatic focusing device and method for microscopic vision system - Google Patents
Automatic focusing device and method for microscopic vision system Download PDFInfo
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- CN110716301B CN110716301B CN201910086635.0A CN201910086635A CN110716301B CN 110716301 B CN110716301 B CN 110716301B CN 201910086635 A CN201910086635 A CN 201910086635A CN 110716301 B CN110716301 B CN 110716301B
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/241—Devices for focusing
Abstract
The invention discloses an automatic focusing device and a method for a microscopic vision system. The micro optical system consists of a micro objective lens, an imaging lens, a condensing lens and a light source. The Z-direction movement device is connected with the collecting lens and drives the collecting lens and the microscope objective to move mutually. The Z-direction movement device is provided with a thimble, and the thimble is closely contacted with the slide and drives the slide to move. The digital camera system carries out microscopic camera shooting on a sample area on the slide in the slide moving process, evaluates the sharpness of the image by utilizing a digital image processing method, determines the best focus and realizes automatic focusing. The invention is suitable for automatic focusing of large numerical aperture microscopic photographing, and has the characteristics of high focusing speed, stable optical resolution, low requirement on flatness of the slice clamping device and the like.
Description
Technical Field
The invention belongs to the field of automatic focusing, and particularly relates to an automatic focusing device and method for a microscopic vision system. The invention can be used for equipment and instruments based on microscopic imaging, and is particularly suitable for rapid focusing of a digital pathological section scanner.
Background
The automatic focusing technology of the microscopic vision system is widely applied in the biomedical field, can improve the speed of digital image shooting, and can prevent the image quality of the acquired microscopic image from being influenced by human factors. Autofocus for conventional microscopic vision systems typically has two ways: one is to drive the objective table to focus, and the other is to drive the objective lens to focus. An automatic focusing microscope based on a liftable automatic stage is disclosed in the ZL200820169109.8 patent, but rapid high-frequency response focusing cannot be realized due to the higher weight of the stage. In patent application 03136023.8 filed by olympus optics corporation of japan, focusing is achieved by moving the imaging lens and the microscope objective lens, and this technique has a high requirement for the movement accuracy of the movement system.
With the development of remote medicine in recent years, remote pathological diagnosis has spawned a new medical device: digital pathological section scanner. A digital pathological section scanner is a system that organically combines a modern digital system with traditional optical principles. The high-resolution digital image for displaying the cell tissue condition of the patient is obtained by collecting and scanning the traditional glass pathological section. And then, the obtained images are automatically spliced and processed in a high-precision multi-view seamless manner by using a computer technology, so that high-quality full-slice digitized images (whole slide image, WSI) are obtained. Once the digital slice images of the patient are generated, medical professionals in different regions can diagnose the patient's condition through the images. The digital slice images of the patients can be transmitted to a remote consultation platform, so that a plurality of consultation specialists can conveniently read slice images of the patients with the difficult and complicated diseases at the same time, and discuss and diagnose the patients.
The automatic focusing technology of the microscopic vision system is a key core technology of the digital pathological section scanner. An automatic focusing device based on a moving object stage and a slice scanning device based on the same are disclosed in 'a digital slice real-time scanning automatic focusing tracking method' (ZL 201310549338.8) and a patent application 201410008180.8 filed by Mike Audi industry group Co. The patent 201410767713.0 'method for scanning pathological section tissues based on rapid and accurate focusing of an image acquisition device' and patent application 201610706704.X 'a tissue section scanning device and a tissue section scanning method' filed by Ningbo Jiang Feng biological information technology Co-Ltd disclose a tissue section scanning technology for moving a microscope objective. The information disclosed by the products in the two markets shows that the digital slice scanning equipment based on the moving microscope objective has higher scanning efficiency. However, techniques based on moving microcomputers have two distinct disadvantages: firstly, the distance between the microscope objective and the imaging lens is changed, and the spatial resolution of the microscope image has difference at different focusing positions, so that the possibility of influencing diagnosis exists; secondly, if the method is to realize efficient scanning, the flatness requirement for slice placement is high, so that the interchangeability of the slice clamp is poor. Therefore, development of a new focusing technique is necessary for further development of digital pathological section scanners.
Disclosure of Invention
The invention aims at providing a novel device and a novel method for realizing automatic focusing of a microscopic vision system by driving a condensing lens according to the application requirement of biomedical field on microscopic image analysis, and the method has the characteristics of high focusing speed, stable spatial resolution, wide slicing adaptability and the like, and is particularly suitable for focusing application of a digital pathological section scanning system.
In order to achieve the purpose, the invention adopts the following technical scheme:
an automatic focusing device for a microscopic vision system comprises a microscopic optical system, a digital camera system, a slide clamping device and a Z-direction movement device;
the micro optical system consists of a micro objective, an imaging lens, a condensing lens and a light source, and the micro objective, the imaging lens and the condensing lens are coaxial with each other; the digital camera system consists of a zoom lens and a camera; the zoom lens is coaxial with the imaging lens; the Z-direction movement device is connected with the collecting lens and drives the collecting lens and the microscope objective to move mutually; the Z-direction movement device is provided with a thimble which is closely contacted with the slide and drives the slide to move.
Further, in the micro optical system of the auto focusing device for the micro vision system, the micro objective lens, the imaging lens and the condenser lens are coaxial with each other.
Further, the Z-direction movement device of the automatic focusing device for the microscopic vision system connects the collecting lens with the movable side through the clamp; the optical axis direction of the condenser lens is parallel to the movement direction of the Z-direction movement device; the optical axis direction of the condenser lens is vertical to the glass slide.
Further, in the automatic focusing device for the microscopic vision system, the ejector pin is arranged on the clamp, and a connecting line between the optical axis center of the collecting lens and the center of the ejector pin is parallel to the long axis of the glass slide.
Further, the automatic focusing device for the microscopic vision system is characterized in that: the upper end face of the clamp is flush with the upper end face of the condenser; the working distance A.W.D. of the condenser, the thickness G.D. of the glass slide and the height H of the ejector pin beyond the upper end face of the condenser have the following relation: h=a.w.d. -g.d.
The working distance O.W.D. of the microscope objective lens of the automatic focusing device for the microscopic vision system has the following relation with the minimum distance MinD from the upper surface of the glass slide to the front lens surface of the microscope objective lens: minD=N×O.W.D., and 0.1< N <0.95; the working distance o.w.d. of the microscope objective has the following relation with the maximum distance MaxD from the upper surface of the slide to the front lens surface of the microscope objective: maxd=m×o.w.d., and 1.2< M <10; the stroke l=maxd-MinD of the Z-direction movement device.
The automatic focusing device for the microscopic vision system is characterized in that: the slide clamping device is provided with a unilateral guiding dovetail groove structure along one side of the long side direction of the slide, and a spring thimble is arranged on the other side; the top end of the spring thimble is a wedge-shaped surface.
Further, the automatic focusing device for the microscopic vision system is characterized in that a slide clamping device is connected with a guide mechanism, and the guide mechanism ensures that a slide moves along the optical axis direction of a microscope objective under the action of a thimble; a pressure spring is arranged between the movable side and the stationary side of the guide mechanism, and the pressure spring applies a force far away from the microscope objective to the movable side; the preferred guide mechanism comprises: the guide rail and the slide block mechanism are parallelogrammic plane link mechanisms.
By means of the device, the invention provides an automatic focusing method for a microscopic vision system, which changes the distance between a sample to be observed and an objective lens to realize focusing by driving a condensing lens to further move the position of a slide, and specifically comprises the following steps:
step 1, controlling a Z-direction movement device to enable a condensing lens to be far away from a microscope objective until the negative limit of the movement device, enabling a slide clamping device to be far away from the microscope objective under the action of a pressure spring, and finally enabling the distance from the upper surface of a slide to the surface of a front lens of the microscope objective to be MaxD;
step 2, controlling a Z-direction movement device to enable a condensing lens to move by a step distance delta d towards the direction close to a microscope objective, enabling a glass slide to also move by a step distance delta d towards the microscope objective under the action of a thimble, controlling a digital camera system to shoot a view field image and recording the current position;
step 3, controlling a Z-direction movement device to enable a condensing lens to move towards a direction close to a microscope objective, shooting a view image every time a step delta d advances in step 2, and recording a corresponding position until the distance from the upper surface of a glass slide to the surface of a front lens of the microscope objective is MinD;
step 4, calculating sharpness of all view images, finding out an image with the largest sharpness, finding out k adjacent images before and after the position, fitting the sharpness and the position according to a Gaussian function, and solving a position Xpos corresponding to a maximum point of the Gaussian function in a section, wherein the position is a focus of the current view, and the specific value of k meets the following conditions:
0.3×O.W.D./Δd≤k≤ 0 .8×O.W.D./Δd；
and 5, controlling the Z-direction movement device to move to Xpos, and completing focusing.
Compared with the prior art, the invention has the beneficial effects that:
first, focusing is achieved by driving the condenser lens and then moving the slide, which has a better illumination effect for a microscope objective with a large numerical aperture (above 0.6). Because the large numerical aperture microscope objective lens often needs a collecting lens with a large numerical aperture (more than 1.1), and the working distance of the collecting lens with the large numerical aperture is often smaller, the collecting lens and the thimble are connected together through a clamp within 1mm from a slice, so that the working distance can be ensured to be stable.
Second, the method for realizing focusing through the top slice provided by the invention has good self-adaptability. Because of the existence of the action of the pressure spring, when the thickness of the tissue on the slide is uniform enough, the thimble can ensure that the tissue is automatically at the focus position without the need of repositioning the position because of poor flatness of the placement of the slice, thereby improving the focusing efficiency, and being particularly suitable for the application of a pathological section scanning system.
Third, the invention does not change the distance among the microscope objective, the imaging lens and the camera, so the spatial resolution of the obtained microscopic image is stable.
Drawings
FIG. 1 is a schematic structural diagram of an auto-focusing apparatus for a microscopic vision system according to the present invention;
FIG. 2 is a schematic diagram of the positional relationship of a slide, ejector pins and a collection lens according to an embodiment of the present invention;
FIG. 3 is a schematic view of a guide rail and slider guide mechanism according to an embodiment of the present invention;
FIG. 4 is a schematic view of a planar link guide in accordance with an embodiment of the present invention;
FIG. 5 is a schematic diagram of solving for focal position using Gaussian function fitting in accordance with an embodiment of the invention.
Detailed Description
The invention will now be described in detail with reference to the drawings and examples.
The embodiment of the invention relates to a novel device and a novel method for realizing automatic focusing of a microscopic vision system by driving a condensing lens, which can be used for focusing application of a digital pathological section scanning system.
As shown in fig. 1, an automatic focusing device for a microscopic vision system includes a microscopic optical system, a digital camera system, a slide holding device 301, and a Z-direction moving device 401, wherein: the micro optical system consists of a micro objective 101, an imaging lens 102, a condenser 103 and a light source 104; the digital camera system consists of a zoom lens 201 and a camera 202; the Z-direction movement device 401 is connected with the condenser 103 and drives the condenser 103 and the microscope objective 101 to move mutually; the Z-direction moving device 401 is provided with a thimble 402, and the thimble 402 is closely contacted with the slide 501 and drives the slide 501 to move.
In this embodiment, the microscope objective 101, the imaging lens 102, and the condenser 103 are coaxial with each other. The variable magnification lens 201 is also coaxial with the imaging lens 102. The magnification-varying mirror 201 preferably has a magnification of 2 times, 1 times, 0.75 times, 0.61 times, 0.5 times, and 0.3 times. When the magnification of the magnification-varying mirror 201 is 1, the magnification-varying mirror 201 may have no physical lenses of a solid body.
In the present embodiment, an auto-focusing device for microscopic vision system, wherein a Z-direction movement device 401 connects a condenser 103 with a movable side of the Z-direction movement device 401 through a clamp 403; the optical axis direction of the condenser 103 is parallel to the movement direction of the Z-direction movement device; the condenser 103 is perpendicular to the slide in the optical axis direction.
As shown in fig. 2, in the present embodiment, an auto-focusing device for a microscopic vision system, a thimble 402 is mounted on a jig 403, and a line between the center of the optical axis of a condenser 103 and the center of the thimble 402 is parallel to the long axis of a slide 501. The upper end surface of the clamp 403 is flush with the upper end surface of the condenser 103; the working distance a.w.d. of the condenser 103, the thickness g.d. of the slide 501, and the height H of the ejector pin 402 above the upper end surface of the condenser 103 have the following relationship: h=a.w.d. -g.d.
In this embodiment, an auto-focusing apparatus for a microscopic vision system has the following relationship between the working distance o.w.d. of the microscope objective 101 and the minimum distance MinD from the upper surface of the slide 501 to the front lens surface of the microscope objective 101: minD=N×O.W.D., and 0.1< N <0.95; the working distance o.w.d. of the micro objective 101 is related to the maximum distance MaxD from the upper surface of the slide 501 to the front lens surface of the micro objective 101 as follows: maxd=m×o.w.d., and 1.2< M <10; the stroke l=maxd-MinD of the Z-direction movement device 401.
As shown in fig. 1, 3 and 4, an automatic focusing device for a microscopic vision system is provided, wherein a slide clamping device 301 is provided with a unilateral guiding dovetail groove structure 304 along one side of a long side direction of a slide 501, and the other side is provided with a spring thimble 303; the top end of the spring pin 303 is a wedge surface.
In this embodiment, an automatic focusing device for a microscopic vision system is provided, in which a slide holding device 301 is connected to a guide mechanism 302, and the guide mechanism 302 ensures that a slide 501 moves along the optical axis direction of a microscope objective 101 under the action of a thimble 402.
This embodiment provides two implementations of the guide mechanism 302.
Implementation one of the guide mechanism 302: as shown in fig. 3, the guide mechanism 302 is composed of a guide rail 306 and a slider 307, where the preferred type of guide rail is a cross roller guide rail. The slide 307 is mounted on the movable side 308 of the guide mechanism 302 and the guide rail 306 is mounted on the stationary side 309 of the guide mechanism 302. A compression spring 305 is provided between the movable side 308 and the stationary side 309 of the guide mechanism 302, the compression spring 305 exerting a force on the movable side 308 away from the microscope objective 101.
Implementation two of the guide mechanism 302: as shown in fig. 4, the guide mechanism 302 is a parallelogram planar linkage. The present embodiment provides an integral planar linkage based on flexible hinges, where the movable side 308 and the stationary side 309 of the guide mechanism 302 are kept connected by a beam 310 and a beam 311, and the movable side 308, the stationary side 309, the beam 310 and the beam 311 have a parallelogram structure. A compression spring 312 and a compression spring 313 are arranged between the movable side 308 and the stationary side 309 of the guide mechanism 302, and the compression spring 312 and the compression spring 313 apply a force to the movable side 308 away from the microscope objective 101.
By means of the device, the invention provides an automatic focusing method for a microscopic vision system, which changes the distance between a sample to be observed and a microscope objective 101 to realize focusing by driving a condensing lens 103 to further move the position of a glass slide 501, and specifically comprises the following steps:
step 1, controlling a Z-direction movement device 401 to enable a condensing lens 103 to be far away from a micro objective lens 101 until a negative limit of the movement device, enabling a slide clamping device 301 to be far away from the micro objective lens 101 under the action of a pressure spring, and finally enabling the distance from the upper surface of a slide 501 to the front lens surface of the micro objective lens 101 to be MaxD;
step 2, controlling the Z-direction movement device 401 to enable the condenser 103 to move by a step distance delta d towards the direction approaching the microscope objective 101, enabling the glass slide 501 to also move by a step distance delta d towards the microscope objective 101 under the action of the ejector pin, controlling the digital camera system to shoot a visual field image and recording the current position;
step 3, controlling the Z-direction movement device 401 to move the collecting lens 103 towards the direction approaching the microscope objective 101, shooting a view image every one step distance deltad and recording the corresponding position as per step distance deltad in step 2 until the distance from the upper surface of the glass slide 501 to the front lens surface of the microscope objective 101 is MinD;
step 4, as shown in fig. 5, calculating sharpness of all view images to obtain a position-sharpness curve 601; in the figure, the x-axis is the motion position of the Z-direction motion device 401 corresponding to the view image, and an image with the maximum sharpness and the corresponding position 603 are found out; the k adjacent images are found before and after the position 603, and the specific value of k meets the following conditions: 0.3 x O.W.D./Δd.ltoreq.k.ltoreq.0.8 x O.W.D./Δd; fitting a position-sharpness curve corresponding to the 2k image according to a Gaussian function, wherein the fitted curve is shown as 602, and solving a position Xpos corresponding to a Gaussian function maximum point in a section, wherein the position is a focus of the current field; a Gaussian function is adopted in fitting.
And 5, controlling the Z-direction movement device 401 to move to Xpos, and completing focusing.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (4)
1. An automatic focusing device for microscopic vision system, includes micro optical system, digital camera system, slide clamping device and Z to motion device, its characterized in that:
the micro optical system consists of a micro objective, an imaging lens, a condensing lens and a light source, and the micro objective, the imaging lens and the condensing lens are coaxial with each other; the digital camera system consists of a zoom lens and a camera; the zoom lens is coaxial with the imaging lens; the Z-direction movement device is connected with the collecting lens and drives the collecting lens and the microscope objective to move mutually; the Z-direction movement device is provided with a thimble which is closely contacted with the slide and drives the slide to move;
the magnification of the variable magnification lens (201) is 2 times, 1 times, 0.75 times, 0.61 times, 0.5 times and 0.3 times;
the Z-direction movement device connects the condensing lens with the movable side of the Z-direction movement device through a clamp; the optical axis direction of the condenser lens is parallel to the movement direction of the Z-direction movement device; the optical axis direction of the condensing lens is vertical to the glass slide;
the ejector pin is arranged on the clamp, and a connecting line between the optical axis center of the collecting lens and the center of the ejector pin is parallel to the long axis of the glass slide; the upper end face of the clamp is flush with the upper end face of the condenser; the working distance A.W.D. of the condenser, the thickness G.D. of the glass slide and the height H of the ejector pin beyond the upper end face of the condenser have the following relation: h=a.w.d. -g.d.;
the working distance o.w.d. of the microscope objective has the following relation with the minimum distance MinD from the upper surface of the slide to the front lens surface of the microscope objective: minD=N×O.W.D., and 0.1< N <0.95; the working distance o.w.d. of the microscope objective has the following relation with the maximum distance MaxD from the upper surface of the slide to the front lens surface of the microscope objective: maxd=m×o.w.d., and 1.2< M <10; the stroke L=MaxD-MinD of the Z-direction movement device;
the slide clamping device is provided with a unilateral guiding dovetail groove structure along one side of the long side direction of the slide, and a spring thimble is arranged on the other side; the top end of the spring thimble is a wedge-shaped surface;
the slide clamping device is connected with the guide mechanism, and the guide mechanism ensures that the slide moves along the optical axis direction of the microscope objective under the action of the ejector pin; a compression spring is arranged between the movable side and the stationary side of the guide mechanism, and the compression spring applies a force to the movable side, which is far away from the microscope objective.
2. An autofocus device for a microscopic vision system as in claim 1, wherein: the guide mechanism is a crossed roller guide rail mechanism and consists of a guide rail and a sliding block, the sliding block is arranged on the moving side of the guide mechanism, and the guide rail is arranged on the fixed side of the guide mechanism; a pressure spring is arranged between the movable side and the stationary side of the guide mechanism, and the pressure spring applies a force to the movable side, which is far away from the microscope objective.
3. An autofocus device for a microscopic vision system as in claim 1, wherein: the guide mechanism is an integrated plane link mechanism based on a flexible hinge, an upper beam and a lower beam are used for keeping connection between a movable side and a stationary side, and the movable side, the stationary side and the lower beam are integrally in a parallelogram structure; and two pressure springs are arranged between the movable side and the stationary side of the guide mechanism, and apply a force far away from the microscope objective to the movable side.
4. A method of implementing an autofocus device for a microscopic vision system as claimed in claim 2 or 3, characterized by: the focusing is realized by changing the distance between the sample to be observed and the objective lens by moving the slide position, and the method specifically comprises the following steps:
step 1, controlling a Z-direction movement device to enable a condensing lens to be far away from a microscope objective until the Z-direction movement device is negatively limited, enabling a slide clamping device to be far away from the microscope objective under the action of a pressure spring, and finally enabling the distance from the upper surface of a slide to the surface of a front lens of the microscope objective to be MaxD;
step 2, controlling a Z-direction movement device to enable a condensing lens to move by a step distance delta d towards the direction close to a microscope objective, enabling a glass slide to also move by a step distance delta d towards the microscope objective under the action of a thimble, controlling a digital camera system to shoot a view field image and recording the current position;
step 3, controlling a Z-direction movement device to enable a condensing lens to move towards a direction close to a microscope objective, shooting a view image every time a step delta d advances in step 2, and recording a corresponding position until the distance from the upper surface of a glass slide to the surface of a front lens of the microscope objective is MinD;
step 4, calculating sharpness of all view images, finding out an image with the largest sharpness, finding out k adjacent images before and after the position of the image, fitting the sharpness and the position according to a Gaussian function, and solving a position Xpos corresponding to a maximum point of the Gaussian function in a section, wherein the position is taken as a focus of the current view, and the specific value of k is 0.3X O.W.D./Deltad is less than or equal to k and less than or equal to 0.8X O.W.D./Deltad;
and 5, controlling the Z-direction movement device to move to Xpos, and completing focusing.
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