JPS6052404B2 - automatic focus adjustment device - Google Patents

Description

[Detailed description of the invention] [Field of application of the invention] The present invention relates to an automatic focus adjustment device for a microscope.

[Background of the Invention] Focus adjustment of a microscope is performed by keeping the optical path length between the sample (subject) and the objective lens constant.

This variation in optical path length is the reason why the microscope image is out of focus, and if this value exceeds the depth of focus of the objective lens (determined by the magnification and numerical aperture of the objective lens, which is usually about ±1.7 μm) The image becomes out of focus. It is generally known that the difference between an out-of-focus image and an in-focus image is noticeable in the density and first derivative of the image. Figure 1 shows an example of this relationship, with the horizontal axis representing the displacement of the optical path length and the vertical axis representing the first-order differential value (absolute value). gradually decreases as the value shifts. Therefore, this differential value can be effectively used as a signal indicating the degree of focus of the image. Although the above-mentioned image density can be used as the signal indicating the degree of focus, an example in which a differential value is used will be explained here. Automatic focus adjustment of a microscope is performed by detecting the maximum value of the differential value just mentioned using a method called the mountain climbing method.

This is done by changing the optical path length between the sample and the objective lens in steps of d (more than the depth of focus) in the direction of increasing the differential value (there are methods such as moving the stage up and down, the lens barrel up and down, and the objective lens up and down, etc.)
Here, we will discuss the upper and lower stages as an example), the optical path length Dl
D-D. ．． Differential value V of D+d. ．． ．． VO-D. ．． VO+
This is a method of finding a position where d satisfies the following relationship. IVO
l>1V0-dl and IVDI>VO+DI Using this method, all you have to do is make sure it's in focus.3
It is necessary to move the stage several times, and a long time is required for automatic single focus adjustment in order to eliminate image deterioration due to afterimages in the imaging system and vibrations of the stage. [Object of the Invention] The present invention provides a high-speed automatic focus adjustment device for a microscope, which eliminates the drawbacks of the above-mentioned automatic focus adjustment.

[Summary of the Invention] First, the properties of the optical system that forms the basis of the present invention will be described.

A microscope consists of an objective lens and an eyepiece.
When the image of the sample is formed on the imaging surface of the imaging system, it is in focus. Now, the optical path length between the objective lens and the sample is +d
, the image of the eyepiece approaches the eyepiece by ΔK. Generally, if the subject moves by a minute value "Δa" with a lens of magnification M, the position of the image will be ΔAM2
only changes. Therefore, if the magnifications of the objective lens and eyepiece are M and n, respectively, ΔK=Dm2n2,
ΔK becomes an extremely large value, and the total magnification changes so much that it cannot be ignored. Here, if a method is adopted in which the eyepiece is moved by ΔL and the imaging surface is fixed, ΔL≧Dm2 and the total magnification does not change much. (d=5μ, n=1 to m=2
0, the variation in total magnification is 1% or less, ΔL>2wR) In other words, if this method is used, it is possible to take in an image whose focus is shifted by d. In other words, an image that is originally out of focus can be captured as an in-focus image.

Therefore, the light from the objective lens is divided into three equal parts, and the optical path length to the eyepiece is L-ΔL. , L, L + ΔL, and if an imaging system is placed in each, each imaging system will have an optical path length of D + d (back focal position) between the objective lens and the sample.
, D (focus point), and D-d (front focus position) are formed.

In this case, as mentioned earlier, the relationship between d and ΔL is determined by the magnification of the objective lens and the eyepiece lens.
When f8, 1@, if d=5μm, ΔL5-2
7! r! It is n. Therefore, by comparing these images, the degree of focus shift can be determined without moving the stage. In other words, the optical path length to the eyepiece is L - ΔL, . L. ．． L+Δ
The differential value of the image of L is expressed as V - △ VL ~ VL + Δ, respectively. When 1, - Δ, and VL + Δ, the focus is on the optical system with a longer optical path to the eyepiece. It's on.

That is, the focal shift in the -d direction, 2VL-Δ, kuL,,〉
When VL+Δ, the optical system whose optical path length to the eyepiece is L is in focus.

When 3VL-Δ>V>VL+Δ, it can be determined that the optical system with the shorter optical path length to the eyepiece is in focus, that is, there is a defocus in the +d direction.

Therefore, in case 1) above, move the sample a certain distance away from the objective lens, and in case 3) bring the sample a certain distance closer to the objective lens, and repeat this until the state 2) above is achieved. A focused image can be obtained in the imaging system via the long L eyepiece.

This adjustment operation is performed each time the field of view of the microscope is moved, but if the first judgment in each field is in condition 2), there is no need to move the sample stage up and down, and focus adjustment can be performed with the minimum stage movement. This can be achieved with The same effect as just described can also be achieved by placing three eyepiece lenses with different focal lengths at appropriate positions.

For example, three eyepieces are placed at positions where the distance from the objective lens to the eyepiece is constant, and D-D. ．． D. ．．
Three types of eyepieces having focal lengths that focus an image of D+d on the imaging surface may be used. Such lenses can be easily created. [Embodiments of the Invention] Next, an embodiment of the present invention will be described regarding a system having three types of eyepiece lenses having different optical path lengths.

FIG. 2 shows a block diagram of a device according to the invention. A sample 3 on a stage 2 illuminated by a light source 1 is magnified by a microscope 4, and the light is divided into three equal parts by an eyepiece tube 6. this is,
This can be easily realized using a half mirror or the like, and the optical path length from the objective lens 5 is L-ΔLlL. ．． Eyepiece lenses 7, 8, and 9 having the same magnification are placed so that L+ΔL, and the images are captured by imaging devices (eg, T cameras) 10, 11, and 12. This imaging operation is performed by a synchronizing signal from the synchronizing signal generating circuit 18. That is, after the stage 2 is moved laterally by the stage drive circuit 17 and the field of view has been changed, a synchronization signal is issued to the imaging devices 10, 11, and 12, and three types of images are captured. Each image signal is differentiated by differentiating circuits 13, 14, and 15. The comparator 16 holds the peak values of the outputs of the differentiating circuits 13, 14, and 15 as +ΔL. Furthermore, at the time when the imaging of each image is completed, the timing signal issued from the synchronization signal generation circuit 18 causes the peak value V to be increased by 1Δ, then V to be increased,
A comparison is made between VL+Δ and the peak value held is reset. As a result of the size comparison, when V is less than Δ and V is <VL+Δ, the focus is on the optical system with a longer optical path length to the eyepiece. i.e. -d
The output of the comparator 16 indicates that there is a focus shift in the direction, and the synchronization signal generation circuit 18 to the stage drive circuit 17
When a timing signal is issued to the stage drive circuit 17
moves the stage 2 away from the objective lens 5 by a certain distance in response to the output of the comparison circuit 16. VL−Δ,〉VL
〉ぃ∆, the microscope has a shorter optical path length to the eyepiece and is in focus. In other words, the output of the comparator 16 indicates that there is a focus shift in the +d direction, and when a timing signal is issued from the synchronization signal generation circuit 18 to the stage drive circuit 17, the stage drive circuit moves the stage 2 to the objective lens by a certain distance. Bring it closer. When the stage movement in the vertical direction is completed by such control, a synchronization signal is issued again from the synchronization signal generation circuit 18 to the imaging devices 10, 11, and 12, and the above-mentioned synchronization is performed until V + - Δ > VL + ΔL is satisfied. The operation is repeated. What has been described above is automatic focus adjustment using three systems of imaging devices.

However, it is not necessarily a good idea to use two systems of imaging devices to take a defocus image (optical path length L±ΔL).
Therefore, as shown in Fig. 3, if an image with optical path length L and an image with optical path length L + ΔL in which half of the image is optical path length and an image with optical path length L - ΔL in the other half are obtained, each optical path is It is possible to compare long images, which is essentially the same as using three systems.

There are two methods to create an image with optical path length L±ΔL within one screen. One is to shorten the effective optical path length by inserting a piece of glass or the like in the middle of the optical path length so that the image is divided into two. Another method is to construct an eyepiece by combining lenses with different focal lengths.

By using the method described above, an image of L±ΔL can be created within one screen, and automatic focus adjustment using the two systems of imaging devices described above becomes possible.

FIG. 4 is a block diagram showing an embodiment of automatic focus adjustment using two systems of imaging devices. The sample 23 on the stage 22 irradiated by the light source 21 is magnified by the microscope 24,
The eyepiece tube 26 divides the light into two equal parts. This can be easily achieved using a half mirror or the like, and the optical path length from the objective lens 25 is L. ．． Eyepiece 2 with the same magnification so that L+ΔL
7 and 28, and images thereof are captured by imaging devices 29 and 30. In front of the eyepiece lens 28, a glass 31 (for example, semicircular optical, flat) having a thickness such that the effective optical path length is LΔΔL is inserted, and the imaging device 30
L<! 1. Make sure that each half of the L-ΔL image is captured. These images are sent to differentiating circuits 32 and 33 to obtain differential values. To explain this in detail, the synchronous signal generation circuit 36
When a synchronization signal is given to the imaging devices 29 and 30 from
30 captures an image of a portion of optical path length L+ΔL, and 29 captures an image of a portion L1 (see FIG. 3) of the image of optical length L. Differential values of the image signals of these parts are output from differentiating circuits 33 and 32, respectively, and are sent to a comparing circuit 34.
Then, the peak values VL-Δ and L1 of the respective differential values are held. When the imaging of the above portion is completed, a timing signal is sent from the synchronization signal generation circuit to the comparison circuit 34, and the comparison circuit 34 determines the peak value V of the differential value of the image with the optical path length L + ΔL and the peak value of the differential value of the image with the optical path length L+ΔL. The peak value VLl of the differential value of the half partial image of the corresponding optical path length L is compared, the result is held, and the peak-held value is reset. Subsequently, the image capturing device 30 captures an image of a portion of the optical path length L−ΔL, and the image capturing device 29 captures an image of the remaining portion L2 of the image of the optical path length L. The peak value of the differential value of the portion of optical path length L−ΔL held during this time, −Δ, and the remaining portion of the image of optical path length L! The peak value V of the differential value of
When the imaging is completed, timing signals are again applied to the synchronization signals 36 to 34, and the values are compared with L2, and these held values are reset again. When the output of the sharpening comparator 34 is 1V and -Δ and VLl and V and 2 and V and +Δ, the focus is on the optical system with a longer optical path length to the eyepiece, that is, in the -d direction. When the focus shift is 2V and -Δ, and V and 1 and V and 2>V and +Δ, the focus is aligned, and 3V and -ΔL>V, and V
When L2>VL+Δ, the determination result is that the optical system with the shorter optical path length to the eyepiece is in focus, that is, there is a focus shift in the +d direction.

Therefore, in the case of 1) and 3), the stage drive circuit 3
5 to move the stage 22, and repeat the above operation until 2) is satisfied. By using this device, high-speed automatic focus adjustment is possible with two systems of imaging devices.

In the above embodiments, an example was given in which the stage was moved, but it goes without saying that other objects such as the objective lens, lens barrel, etc. may also be moved.

〔Effect of the invention〕

As detailed above, according to the present invention, if the focus is in focus, the focus can be adjusted without moving the stage, and even if the focus is not in focus, the focus can be adjusted with a minimum amount of stage movement, making extremely high-speed focus adjustment possible. becomes.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the path length displacement between the sample and the objective lens, that is, the degree of defocus, and the change in the first-order differential value. FIG. 2 is a diagram showing an embodiment of the present invention. FIG. FIG. 4, which is a diagram for explaining the present invention, is a diagram showing another embodiment of the present invention.

Claims (1)

[Scope of Claims] 1. One objective lens, means for dividing the optical path from the objective lens into three optical paths, and three eyepieces provided in the three optical paths and having different optical path lengths to the objective lens. A microscope that simultaneously obtains three types of optical images of the same subject using these lenses: focal position, front focal position, and back focal position, and a microscope that captures the three types of optical images and converts them into electrical signals. three systems of imaging means, and means for obtaining first, second, and third differential values respectively indicating the degree of defocus of the three types of optical images from the electric signals;
A first differential value indicating the degree of defocus of the optical image at the focal position is combined with a second differential value indicating the degree of defocus of the optical image at the front focal position and a second differential value representing the degree of defocus of the optical image at the rear focal position. a means for comparing with each of the third differential values indicating the degree; and an output of the comparison means to determine whether the first differential value
An automatic focus adjustment device for a microscope, comprising means for controlling the distance of the objective lens to the subject so that it becomes larger than a third differential value. 2. One objective lens, means for dividing the optical path from the objective lens into two optical paths, and two eyepiece lenses provided in the two optical paths and having different optical path lengths to the objective lens, Obtain an optical image at the focal position of the subject using the lens and one of the eyepieces, and partially change the effective optical path length between both lenses using the objective lens and the other eyepiece to obtain an optical image of the subject at the same focal position. a microscope that obtains an optical image at the front focal position and a back focal position within a field of view; a first means for capturing the optical image at the focal position and converting it into an electrical signal; and an optical image at the front focal position and the rear focal position. a second means for imaging and converting the image into an electrical signal; and a second means for imaging and converting the image into an electrical signal; and a second means for imaging and converting the image into an electrical signal; means for determining a first differential value indicating the degree of defocus of a portion corresponding to the optical image at the back focal position; and means for determining the front focal position based on the electric signal from the second means. and means for determining third and fourth differential values respectively indicating the degree of defocus of the optical image at the back focal position; and the first differential value, the third differential value, and the second differential value. means for comparing the fourth differential value and the fourth differential value, respectively;
and means for controlling the distance of the objective lens to the subject so that the first and second differential values become larger than the third and fourth differential values, respectively, based on the output of the comparison means. Features an automatic focus adjustment device for microscopes.