Auto-focus device

Abstract

[Problem] The purpose of the present invention is to provide an autofocus device which is smaller than a galvano mirror and can maintain high focus accuracy even when a mirror with a movable angle is used. Another purpose is to provide an autofocus device which is made compact by simplifying a mirror. [Solution] An autofocus device of the present invention is characterized in that: an angle-variable mirror is arranged on an optical path from a light source to the sample; a shielding plate is arranged on an optical path between the light source and the mirror; a light beam emitted from the light source is reflected by the mirror having a different angle and radiated onto the sample after passing through the shielding plate; an AF camera acquires an optical image of the shielding plate at a different angle of the mirror, the optical image being formed by reflected light from the sample; and a control unit determines a focus state from the optical image of the shielding plate at a different angle of the mirror, and outputs a command signal of a moving operation to a distance adjustment mechanism.

Description

Autofocus Device

[0001] The present invention relates to an autofocusing technique that can reduce the size of an autofocusing device equipped in an optical device and change the offset of the focus position with high precision.

[0002] In recent years, microscopes equipped with autofocus devices that automate the focusing operation performed by humans, similar to cameras and other optical devices, have been developed and are now being used in life science research and quality control in industrial fields.

[0003] In modern life science research, the reduction in computational costs using computers has made it possible to process large amounts of data for screening and statistical analysis. This has led to a growing need for automation of microscopes to observe a large number of samples and acquire large amounts of data.

[0004] Autofocus is one of the key technologies in automating microscopes. There are two main autofocus methods for optical microscopes:

[0005] The first method is the contrast method. In this method, the objective lens or stage is moved in the optical axis direction, the actual image formed is captured by an image sensor, and the focus is scanned while the sharpness of the image is evaluated by image processing. The position where the image sharpness is maximum is the in-focus position. In this method, scanning in the focus direction is required, which is problematic because it takes time to achieve focus. In addition, the sample is exposed to a large amount of light, which can cause photobleaching during fluorescence observation.

[0006] The second method is the phase-contrast method, which involves irradiating the stage with near-infrared light from one direction, and using the reflected light to form an image of the aperture on the imaging element. Based on the relative distance, the system determines whether the current focus state is back-focused (when the focus is on the far side of the sample) or front-focused (when the focus is on the near side of the sample), and then drives a distance adjustment mechanism that moves the objective lens or stage in the focusing direction, i.e., along the optical axis. This method eliminates the need for scanning, and because near-infrared light is used, there is no problem with bleaching. Therefore, microscopes equipped with this type of autofocus device are often used in life science research.

[0007] The second method is primarily used in life science research, but there is a problem with the focus going out over time, so photographs are currently taken by manually fine-tuning the autofocus device. On the other hand, there is a shortage of experimenters to acquire data in research and development in the life science field, so there is a high demand in the field for the automation of microscope photography.

[0008] In the field of life sciences, microscope automation requires high-speed, high-precision focusing, so phase-contrast imaging, which does not require scanning of the objective lens or stage, is increasingly being adopted. However, as mentioned above, phase-contrast imaging suffers from the problem that, after a long time has passed since focusing, the focal point, which is normally on the glass surface, may shift away from the glass surface due to misalignment or distortion of the optical system, resulting in a shift from the target object being observed. In confocal microscopes and fluorescence microscopes, the focal position is within the range of the optical axis, i.e., when the observation target is an entire cell with a certain thickness, a shift in focus may be tolerated as long as the focal position is within the thickness of the cell. However, when observing a glass surface, as with total internal reflection microscopes, which are a type of super-resolution microscope, a shift in the optical axis from the glass surface can cause the image to become blurred, making the captured image unusable for observation. As mentioned above, the automation of microscopic imaging in super-resolution microscopes has not progressed particularly well, and imaging is still performed by manually adjusting the focus. Furthermore, since it is not possible to maintain high focusing accuracy over long periods of time during autofocus operation, it is difficult to achieve long-term time-lapse imaging using super-resolution microscopes.

[0009] Japanese Patent Application Laid-Open No. 2003-144999 discloses a prior art phase difference method that addresses the above-mentioned problems.

[0010] WO2019 / 159627A1

[0011] In conventional autofocus technology, light used for focusing is incident from one direction, and the position of the aperture image detected by the image sensor is controlled so that it is centered. However, with this method, it is difficult to maintain a focused state for a long period of time because the aperture position changes due to misalignment or distortion of the optical system.

[0012] Patent Document 1 discloses a phase-difference autofocus technology developed to address the problems of the prior art. Patent Document 1 first adjusts the angle of autofocus light (hereinafter referred to as AF light) incident on the aperture from two directions, acquires an image (hereinafter referred to as aperture image) formed when each AF light irradiates the sample and passes through the aperture, and measures the position of the aperture image from the image. The difference between the image positions of each aperture image is then calculated, and the stage is moved and controlled in the Z direction (optical axis direction) so that this difference remains constant. If the optical system is distorted, the position of the aperture image in each image will change in the same way. Therefore, as long as there is no optical misalignment or distortion between the two images, the difference in the relative positions of the aperture images will not change. Therefore, this technology can be said to be capable of focusing on a glass surface even after a long period of time. However, Patent Document 1 uses a galvanometer mirror to adjust the angle of the AF light multiple times and capture the aperture image, which requires a mechanism to move the galvanometer mirror. As a result, the device became bulky and required a high-power driver to control the galvanometer mirror, posing challenges to miniaturization and cost reduction.

[0013] Furthermore, when using galvanometer mirror 310, a slit is placed immediately after galvanometer mirror 310 on the optical path, and an optical image of the slit is captured by an autofocus camera. However, if the angle of the galvanometer mirror 310 is changed and the change in the angle of the light beam is too large, some of the light beam will not be able to pass through the slit in the shielding plate 320 (FIG. 7). Specifically, if the radius of the slit is r, the distance between galvanometer mirror 310 and the slit is l, and the angle between the optical axis and the vertical direction is θ, then if r<x=l tan θ, the laser will not be able to pass through the slit. With a large mirror size like galvanometer mirror 310, the diameter of the light beam can be increased to about 1 cm, and all of the light beam will not escape from the slit.

[0014] However, if the mirror size of the galvanometer mirror 310 is reduced in order to miniaturize the autofocus device, the diameter of the laser must also be reduced, and if the angle of the light beam is increased, there is a possibility that the light will completely miss the slit 320 in the shielding plate, which poses a problem that there is a limit to the extent to which the device can be miniaturized (Figure 8).

[0015] To address the above situation, two convex lenses, lens A 330 and lens B 332, are combined between the galvanometer mirror 310 and the slit, making the galvanometer mirror 310 and the slit conjugate, so that even if the angle of the galvanometer mirror 310 is changed arbitrarily, the light beam can always pass through the center of the slit in the shielding plate 320, completely eliminating loss of light quantity (FIG. 9). However, this requires that the two lenses be positioned at a distance on the optical axis, which causes the optical system of the autofocus device to be configured to expand horizontally, which poses a problem of enlargement.

[0016] A second problem with the autofocus method of Patent Document 1 is the issue of offset adjustment. To reduce the size of the entire observation device, it is necessary to mount the imaging lens inside the microscope, which makes it difficult to change the position of the imaging lens with an add-on autofocus device. Therefore, when the imaging lens is placed inside the microscope, it is used in a fixed state. When the imaging lens is fixed, in order to set the focus position on the glass surface, calibration is required at the time of installation to manually adjust the position of the imaging lens without mounting the autofocus device.

[0017] Even if there is some space inside the microscope and the offset can be adjusted by electrically changing the position of the imaging lens, there is still the problem of a small focus range. The focus offset distance fx is given by fx = dx / M, where dx is the displacement of the imaging lens and M is the magnification. ２This can be expressed by the following formula. If we substitute a magnification M of 100x and a displacement dx of 10 mm into the above formula, we can see that the focus offset distance can only be adjusted by about 1 μm. Considering the thickness of the cell, the focus offset distance needs to be adjusted by 5 μm, but if the range is changed to 5x, the imaging lens also needs to be moved 50 mm, which is 5 times the distance. If the moving distance of the imaging lens is longer, the device becomes larger and the time required to move the imaging lens also becomes longer, resulting in the problem of longer time until focus is completed.

[0018] In the method of Patent Document 1, in addition to moving the imaging lens, the focal position can also be changed by changing the convergence position of the difference between the slit positions acquired from two directions. For example, suppose that the focus is on the glass surface when autofocus is performed so that the difference between the centers of gravity of the slits photographed from two directions is 0 (zero). If this difference between the centers of gravity is made non-zero, the focal position can be moved forward or backward from the glass surface. In this case, there is no need to move the imaging lens, but the slit image appears clear when the difference between the centers of gravity is 0, but becomes blurred when it moves away from 0. This blurring reduces the precision of the focus. Therefore, autofocus is basically forced to be performed at a position where the slit is in focus.

[0019] The present invention has been made in consideration of the above-mentioned problems, and aims to provide an autofocus device for an optical device that is even smaller than a galvanometer mirror and can maintain high focusing accuracy even when using a mirror whose angle is adjustable. It also aims to provide an autofocus device for an optical device that is compact due to a simplified mirror control circuit. It also aims to provide an autofocus device for an optical device that allows for a larger offset amount in the focusing direction than conventional autofocus devices.

[0020] In order to solve the above problems, the autofocus device of the present invention is an autofocus device that automatically adjusts the distance between a sample to be observed and an objective lens to achieve focus, and is characterized in that an angle-adjustable mirror is placed on the optical path from a light source to the sample, and a shielding plate is placed on the optical path between the light source and the mirror, and after light emitted from the light source passes through the shielding plate, it is reflected by the mirrors at different angles and is irradiated onto the sample, and an autofocus imaging element acquires light images of the shielding plates with different mirror angles formed by the light reflected from the sample, and a control unit determines the focus state from the light images of the shielding plates with different mirror angles and outputs a command signal for a movement operation to a distance adjustment mechanism.

[0021] The autofocus device of the present invention is also characterized in that a first lens is disposed between the shielding plate and the mirror, and projects an image of the shielding plate onto a central portion of the mirror.

[0022] The autofocus device of the present invention is characterized in that an imaging lens is disposed after the mirror on the optical path, and the imaging lens is movable in the direction of the optical axis.

[0023] The autofocus device of the present invention is also characterized in that at least one second lens is arranged on the optical path between the mirror and the objective lens, or between the objective lens and the autofocus image sensor, and either the imaging lens or the second lens is movable in the optical axis direction.

[0024] The microscope of the present invention is characterized by having the above-mentioned autofocus device.

[0025] The autofocus device of the present invention achieves the advantage of being significantly smaller in size than a galvanometer mirror while maintaining high focusing accuracy by using a MEMS mirror. Furthermore, by disposing a shielding plate with a slit in front of the MEMS mirror, it is possible to prevent light rays from escaping the MEMS mirror and becoming stray light. Furthermore, because the MEMS mirror can be driven with low power and can be powered by a USB, it does not require a high-power control device like a galvanometer mirror.

[0026] According to the autofocus device of the present invention, by arranging a convex lens between the shielding plate and the MEMS mirror in the direction of the optical axis, or more preferably by arranging two convex lenses in combination, the MEMS mirror and the slit can be made conjugate, light rays can be focused at the center of the micro-sized MEMS mirror, and there is an effect that the light intensity is not reduced depending on the angle of the MEMS mirror. Furthermore, because the two convex lenses between the slit and the MEMS mirror can be arranged in the direction of the optical axis toward the sample surface, the optical system does not have to be configured to expand horizontally, and there is an effect that the device does not become bulky.

[0027] Furthermore, according to the autofocus device of the present invention, by arranging a concave lens and a convex lens in that order from the MEMS mirror side between the MEMS mirror and the imaging lens, it is possible to achieve the effect of widening the range of focus offset without significantly moving the position of the imaging lens in the optical axis direction.

[0028] The microscope of the present invention is equipped with an autofocus device using a MEMS mirror, which has the effect of significantly reducing the size of the microscope. Furthermore, a microscope equipped with the autofocus device of the present invention is smaller than conventional microscopes, yet maintains high-precision focus and has a wide focus offset range.

[0029] FIG. 1 is a conceptual diagram showing the basic configuration of the optical system of the autofocus device 1 of the present invention. FIG. 1 is a conceptual diagram showing the optical path before and after the small movable mirror 30 of the autofocus device 1 of the present invention. FIG. 2 is a conceptual diagram showing the basic configuration of the optical system of the autofocus device 2, which is one embodiment of the present invention. FIG. 3 is a conceptual diagram showing the optical path before and after the small movable mirror 30 of the autofocus device 2, which is one embodiment of the present invention. FIG. 4 is a conceptual diagram showing the basic configuration of the optical system of the autofocus device 3, which is another embodiment of the present invention. FIG. 5 is a conceptual diagram showing the offset adjustment operation of the autofocus device 3, which is another embodiment of the present invention. FIG. 6 is a conceptual diagram showing the optical path before and after the galvanometer mirror 310 of a conventional autofocus device. FIG. 7 is a conceptual diagram showing the problem when the galvanometer mirror 310 is replaced with a small movable mirror 312 in a conventional autofocus device. FIG. 8 is a conceptual diagram showing an optical system that prevents loss of light intensity when the galvanometer mirror 310 is used in a conventional autofocus device.

[0030] An embodiment of an autofocus device according to the present invention will be described with reference to the drawings. FIG. 1 is a conceptual diagram showing the basic configuration of the optical system of an autofocus device 1 according to the present invention. In FIG. 1, the autofocus device 1 comprises a light source 10, a slit-equipped shielding plate 20, a small movable mirror 30, a polarizing beam splitter 40, an AF imaging lens 50 (hereinafter referred to as the imaging lens 50), a 1 / λ wavelength plate 60, a dichroic mirror 70, and an autofocus camera 80 (hereinafter referred to as the AF camera 80) as an autofocus image sensor. The light source 10 in FIG. 1 is a point light source. Therefore, the light source 10 is preferably a laser light source 10 with high directionality. A light ray R emitted from the point light source is shown by a solid line (the same applies hereinafter).

[0031] With the above-described configuration, the AF light R emitted from the light source 10 first passes through the slit in the shielding plate 20, and then the small movable mirror 30 adjusts the angle of the AF light R so that it is incident from two directions. The AF light R then passes through the polarizing beam splitter 40, the imaging lens 50, the λ / 4 wavelength plate 60, the dichroic mirror 70, and the objective lens 210, and is then irradiated onto the sample (sample glass 220). The AF light R reflected from the sample at different angles is captured by the AF camera 80, and an image formed by passing through the slit (hereinafter referred to as the slit image) is obtained. The position of the slit image is measured from this image. The difference between the image positions of the individual slit images is then calculated, and the stage is controlled by moving in the Z direction (optical axis direction) so that the difference remains constant.

[0032] It is preferable to use a MEMS mirror as the small movable mirror 30. Unlike a galvanometer mirror, which uses a motor to change the angle of the mirror, the MEMS mirror 30 changes its angle by twisting the mirror supported on an axis using the Lorentz force generated by passing a current through an area where a magnetic field is generated. Therefore, in addition to the small size of the mirror body, the structure for generating the Lorentz force is simple, so it can be driven with low power consumption. For example, it can be operated using a power source from a USB (Universal Serial Bus).

[0033] Furthermore, the diameter of the MEMS mirror 30 is about 1 / 10 the size of a galvanometer mirror. Therefore, compared to when a galvanometer mirror is used, the autofocus device 1 can be made extremely small. Hereinafter, the small movable mirror 30 will be referred to as the MEMS mirror 30.

[0034] However, as shown in Figure 8, in the conventional technology, when the movable mirror is changed to a small movable mirror 312, in order to significantly change the angle of the AF light R, when the small movable mirror 312 is rotated so that the angle of the AF light R relative to the direction perpendicular to the optical axis becomes large, there is a case where the AF light R completely deviates from the slit.

[0035] Furthermore, in the case of the MEMS mirror 30, the mirror has a small diameter of only a few millimeters, and the electronic circuit for operating the mirror (hereinafter referred to as the "drive circuit") is exposed. Therefore, in the conventional method of Patent Document 1, the laser light (AF light R) hits the drive circuit portion for changing the angle of the MEMS mirror 30, which is provided around the MEMS mirror 30. As shown in FIG. 8, when the AF light R hits the drive circuit portion of the small movable mirror 312, it is diffusely reflected and becomes stray light SL, which causes the slit image to become unclear. The stray light SL reduces the precision and accuracy of autofocus. A configuration for solving the above problem will be described below.

[0036] Unlike conventional technology, the autofocus device 1 of the present invention is configured such that a shielding plate 20 with a slit is placed between the laser light source 10 and the MEMS mirror 30, and no shielding plate with a slit is placed after the MEMS mirror 30. In the present invention, the shielding plate 20 is placed before the MEMS mirror 30 to reduce the AF light R that strikes the drive circuit parts other than the mirror. By making the slit diameter 1 mm or less, the AF light R can be contained above the mirror, and stray light SL can be suppressed.

[0037] Furthermore, since no shielding plate with a slit is placed downstream of the MEMS mirror 30, there is no problem of the AF light R straying from the slit when the MEMS mirror 30 is rotated so that the angle of the AF light R relative to the direction perpendicular to the optical axis becomes large.

[0038] 2 is a conceptual diagram showing the optical path before and after the small movable mirror (MEMS mirror) 30 of the autofocus device 1 of the present invention. Because the AF light R spreads due to diffraction when passing through the slit, it is preferable to set the distance between the shielding plate 20 and the mirror extremely short, even if the AF light is a highly directional laser light.

[0039] FIG. 3 is a conceptual diagram showing the basic configuration of the optical system of an autofocus device 2 according to an embodiment of the present invention. In addition to the configuration shown in FIG. 1 , the autofocus device 2 of the present invention has a configuration in which a first lens is disposed between the occlusion plate 20 and the MEMS mirror 30. FIG. 4 is a conceptual diagram showing the optical path before and after the small movable mirror (MEMS mirror) 30 of the autofocus device 2 of the present invention. By disposing the first lens between the occlusion plate 20 and the MEMS mirror 30, an image of the occlusion plate 20 can be formed on the MEMS mirror 30. Although it is possible to form an image on the MEMS mirror 30 using a single first lens, by using two lenses, lens a 90 and lens b 92, as shown in FIGS. 3 and 4 , it is possible to form a slit image of the occlusion plate 20 on the MEMS mirror 30 and to direct the light in a straight line, thereby allowing the MEMS mirror 30 and the occlusion plate 20 to overlap, i.e., the distance between the MEMS mirror 30 and the occlusion plate 20 to be optically zero. This reduces the influence of spreading due to diffraction of the AF light R at the shielding plate 20, and enables a clear slit image to be obtained without the risk of the light intensity decreasing depending on the angle of the MEMS mirror 30. As a result, the shape of the obtained slit image remains the same even if the angle of the MEMS mirror 30 is changed, and the effort required for offset adjustment during autofocus can be reduced.

[0040] By using two lenses, the distance in the optical axis direction in autofocus device 2 becomes longer, but the size does not increase in the direction perpendicular to the optical axis. Even when comparing autofocus device 2 according to the present invention in Figure 3 with autofocus device 1 according to the present invention shown in Figure 1, the size of the device does not change significantly because AF camera 80 is positioned parallel to the two lenses.

[0041] Furthermore, the embodiment of the autofocus device of the present invention is configured to be able to adjust (offset) the focus position. By being able to control the movement of the imaging lens 50 back and forth along the optical axis, the focus position can be offset. The offset distance fx of this focus position is given by fx = dx / M, where dx is the displacement of the imaging lens 50 and M is the magnification. ２ This becomes:

[0042] By adding a second lens in addition to the imaging lens 50 and controlling the movement of any one of the lenses in the optical axis direction, the focus offset range can be widened.

[0043] FIG. 5 is a conceptual diagram showing the basic configuration of the optical system of the autofocus device 3 of the present invention, which can expand the offset range of the focus position in the optical axis direction. FIG. 6 is a conceptual diagram showing the offset adjustment operation of the autofocus device 3 of the present invention. The second lens may be a single convex or concave lens. However, by arranging a concave lens 94 and a convex lens 96 in this order on the optical path of the AF light R between the polarizing beam splitter 40 and the imaging lens 50, the concave lens 94 and the convex lens 96 function as a Galilean expander. That is, when the laser AF light R passes through, the beam diameter expands or contracts, but the angle of the light beam remains unchanged. By moving either the concave lens 94 or the convex lens 96 in the optical axis direction of the AF light R, the focus position is offset and changed in the optical axis direction. When parallel light such as a laser is introduced into the expander, the focal point is at infinity. Moving the lens eliminates the parallel light, and the focal point becomes finite. This means that a small movement of the lens can significantly change the focal position.

[0044] 6 is one example, and the same effect can be obtained even if the second lens is placed on the optical path between the MEMS mirror 30 and the objective lens 210, or between the objective lens 210 and the AF camera 80, as shown in Fig. 5. However, if the second lens is placed inside the microscope, it becomes difficult to move the second lens, so it is preferable to place it inside the autofocus device 3.

[0045] When using the imaging lens 50 alone, the focus position changes linearly in the optical axis direction with respect to the displacement dx of the imaging lens 50. However, when using the concave lens 94 and the convex lens 96, the focus position changes nonlinearly in the optical axis direction. Therefore, by adjusting the arrangement of the concave lens 94 and the convex lens 96 when attaching the autofocus device 3 to, for example, a microscope, it is possible to increase the offset distance fx of the focus position with a small amount of displacement dx. In other words, the offset range can be widened. However, because the amount of change in offset is not linear, when changing the focus position at regular intervals, it is necessary to measure the amount of change in offset before performing the autofocus operation, understand the correspondence between the displacement dx of the imaging lens 50 and the offset distance fx of the focus position, and then perform calibration.

[0046] The autofocus device of the present invention is particularly effective in the field of life science research. Compared to conventional autofocus devices, the autofocus device of the present invention is significantly smaller and less expensive, making it easier to implement. This allows for easy automation of microscopes, making it easier to observe a large number of samples, process large amounts of data, and perform screening and statistical analysis.

[0047] REFERENCE SIGNS LIST 1 autofocus device 2 autofocus device 3 autofocus device 10 light source (laser light source) 20 shielding plate 30 small movable mirror (MEMS mirror) 40 polarizing beam splitter 50 imaging lens 60 λ / 4 wave plate 70 dichroic mirror 80 autofocus camera (AF camera) 90 lens a 92 lens b 94 concave lens 96 convex lens 210 objective lens 220 sample glass 310 galvanometer mirror 312 small movable mirror 320 shielding plate 330 lens A 332 lens B R AF light SL stray light

Claims

1. An autofocus device that automatically adjusts the distance between a sample to be observed and an objective lens to achieve focus, the autofocus device comprising: a mirror whose angle can be changed, positioned on the optical path from a light source to the sample; a shielding plate, positioned on the optical path between the light source and the mirror; light emitted from the light source passes through the shielding plate, is reflected by the mirrors with different angles, and is irradiated onto the sample; an autofocus image sensor acquires optical images of the shielding plates with different mirror angles formed by light reflected from the sample; and a control unit determines the focus state from the optical images of the shielding plates with different mirror angles and outputs a command signal to a distance adjustment mechanism to perform a movement operation.

2. An autofocus device according to claim 1, characterized in that a first lens is disposed between the shielding plate and the mirror, and projects an image of the shielding plate onto a central portion of the mirror.

3. An autofocus device according to claim 1 or claim 2, characterized in that an imaging lens is arranged after the mirror on the optical path, and the imaging lens is movable in the direction of the optical axis.

4. The autofocus device described in claim 3, characterized in that at least one second lens is placed on the optical path between the mirror and the objective lens, or between the objective lens and the autofocus image sensor, and either the imaging lens or the second lens can move in the direction of the optical axis.

5. A microscope comprising the autofocus device according to claim 1.

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