JP2013235110A - Autofocus device and microscope including the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an autofocus device that enables a stable, accurate and quick focus adjustment, and to provide a microscope including the autofocus device.SOLUTION: The autofocus device comprises: an objective lens system 11 that condenses a light flux from a specimen S; a structuring pupil 14 that includes a plate-shape member 14a provided at a position almost conjugate to a position of an exit pupil position of the objective lens system 11 and blocking the light flux from the objective lens system 11, a plurality of apertures 14h of the plate-shape member 14a and filters 14r, 14b and 14g attached to the plurality of apertures 14h and transmitting only predetermined color components; an imaging lens system 15 that images a light flux transmitted through each filter on an imaging element 16; and a focus position detection part 17 that processes and analyzes image data acquired from the first imaging element 16 and detects a focus position. The focus position detection part 17 acquires from the first imaging element 16 an image based on the light flux transmitted through each filter and separated into a plurality of different color components and detects a focus position of the objective lens system 11 from how these images are overlapped.

Description

  The present invention relates to an autofocus device.

  As an autofocus (hereinafter referred to as AF) device for a microscope, an active method for projecting a specimen from an AF light source, detecting a reflected light from the specimen, calculating a defocus amount, and controlling the position of the objective lens (For example, refer to Patent Document 1) and a passive method (for example, refer to Patent Document 2) that detects the amount of defocus by detecting light from a specimen and performs focusing.

Japanese Patent Laid-Open No. 2003-167183 Japanese Patent Laid-Open No. 5-21318

  Regardless of the method, it is desirable for the autofocus device to perform focus adjustment stably, accurately, and quickly.

  The present invention has been made in view of such problems, and an object of the present invention is to provide an autofocus device capable of performing focus adjustment stably, accurately and quickly, and a microscope having the same.

  In order to achieve such an object, according to a first aspect illustrating the present invention, an objective lens system that collects a light beam from a specimen, and an exit of the objective lens system on the exit side of the objective lens system A plate-like member provided at a pupil position or a position substantially conjugated with the pupil position, which shields the light beam from the objective lens system, a plurality of openings formed in the plate-like member, and a plurality of openings attached A structured pupil having a filter that transmits only a light beam of a predetermined color component, an imaging lens system that forms an image on the image sensor with a light beam that has passed through each filter of the structured pupil, and an image acquired from the image sensor A focal position detector that processes and analyzes data and detects a focal position of the objective lens system, and the focal position detector transmits a plurality of filters of the structured pupil from the imaging device. Split into different color components Image acquires based on the light beam, the overlapping state of an image based on the light beam of the plurality of different color components, said autofocus apparatus and detecting the focal position of the objective lens system is provided.

  Each lens system may be constituted by one lens or may be constituted by a plurality of lenses.

  Moreover, according to the 2nd aspect which illustrates this invention, the microscope characterized by having the autofocus apparatus of a 1st aspect is provided.

  According to the present invention, it is possible to provide an autofocus device capable of performing focus adjustment stably, accurately and quickly, and a microscope having the same.

1 is a configuration diagram of an autofocus device according to a first embodiment and a microscope in which the device is incorporated. FIG. It is a figure explaining the principle of color shift. (A) is a plan view of the structured pupil, (b) is a schematic diagram of an image captured by the first image sensor when there is no focus shift, and (c) is the first image sensor when there is a focus shift. It is a schematic diagram of the image imaged by. It is a figure explaining the relationship between a reference image, the sample image based on the light beam of each color component, and a local window. It is a figure explaining the calculation method of the amount of color shift. It is a figure explaining the calculation method of the direction of a focus gap. It is a flowchart which shows operation | movement of an auto-focus apparatus. It is a block diagram of another microscope based on 1st Embodiment and the microscope incorporating this apparatus. It is a figure explaining the liquid crystal display element of a pixel structure, and the liquid crystal display element in which the modulation | alteration part is formed in the ring shape. It is a block diagram of the autofocus apparatus which concerns on 2nd Embodiment, and the microscope in which this apparatus is incorporated. It is a figure explaining the calculation method of the tilt information of a sample surface. It is a figure which shows the structural example of a three-dimensional correction mechanism.

  Hereinafter, embodiments according to the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 shows a configuration example of a microscope in which the autofocus device according to the first embodiment is incorporated. In the first embodiment, a passive method that does not project the focus index light is employed as the autofocus device. In this method, the captured sample image is used as it is as an autofocus index in real time. This method has an advantage that information on the positional deviation between the image formed by the objective lens system and the original image plane, that is, information on the focal deviation can be acquired at a high speed by a very simple method.

  First, the configuration of the microscope will be described with reference to FIGS. 1 (a) and 1 (b). As shown in FIG. 1A, the microscope includes an objective lens system 11, a pupil magnifying lens system 12, a beam splitter 13, and a second imaging lens system 31 arranged in order along the optical axis from the specimen side. An observation optical system and a second image sensor 32 are included. When the sample S is in focus with respect to the objective lens system 11, the light beam from the sample S sequentially passes through the objective lens system 11, the pupil enlarging lens system 12, and the beam splitter 13, and the second An image is formed on the imaging surface of the second imaging element 32 by the imaging lens system 31. Note that FIG. 1 shows a case where a focus shift has already occurred. Therefore, it should be noted that the sample S and the first image sensor 16 and the second image sensor 32 are not in a conjugate relationship.

  On the imaging surface of the second imaging element 32, the way in which the image of the sample S is formed differs depending on the in-focus state. For example, if the objective lens system 11 is in the in-focus position, as shown in the left diagram of FIG. 1B, a clear image of the specimen S is formed, and if the objective lens system 11 is not in the in-focus position, FIG. As shown in the right figure of 1 (b), an image of the blurred specimen S is formed.

  Next, the autofocus device will be described with reference to FIGS. 1 (a) and 1 (c). As shown in FIG. 1 (a), the autofocus device includes an objective lens system 11, a pupil magnifying lens system 12, a beam splitter 13, a structured pupil 14, and a first pupil arranged in order from the sample side along the optical axis. An AF optical system including an imaging lens system 15 and a first image sensor 16 are included. In order to achieve an ideal optical system, the lens system is configured to be telecentric on both sides, but this is not an absolute requirement.

  The objective lens system 11 collects the light from the sample S and forms an image of the sample S. The objective lens system 11 is configured to be reciprocally movable in a direction along the optical axis by a driving unit 19 described later, and the relative position in the optical axis direction between the objective lens system 11 and the sample S can be changed. ing.

  The pupil magnifying lens system 12 enlarges and relays the exit pupil of the objective lens system 11.

  The beam splitter 13 separates the light from the sample S that has passed through the pupil enlarging lens system 12 into two parts, leads one to the structured pupil 14 of the AF optical system, and the other to the second of the observation optical system. Guide to the imaging lens 31.

  The structured pupil 14 is provided at a position substantially conjugate with the exit pupil position of the objective lens system 11 on the exit side of the objective lens system 11, a plate-like member 14 a that shields the light beam from the objective lens system 11, and a plate-like shape A plurality of openings 14h formed in the member 14a and filters that are attached to the plurality of openings 14h and transmit only light beams of a predetermined color component, for example, red, green, and blue color filters 14r, 14g, and 14b are provided. The plurality of openings 14 h are provided at positions that are decentered with respect to the center of the exit pupil of the objective lens system 11.

  In the present embodiment, all three colors of red, green, and blue are assumed as filters below, but this method can be realized if there are two or more colors. Further, the transmission wavelength region of each filter may be a measurable band of the first image sensor 16 and is not limited to a specific wavelength. The installation position of the structured pupil 14 is not limited to the above, and may be the pupil position of the objective lens system 11. The structure of the structured pupil 14 is not limited to the present embodiment as long as it can acquire an image for each color component with high accuracy. However, if the opening 14h is too large, the focus detection accuracy will be lowered as will be described later, so care must be taken. The structured pupil 14 may be provided with a fixed aperture in advance, or may be electrically generated by a spatial light modulator or the like.

  The first imaging lens system 15 causes the first imaging element 16 to form an image of the light flux that has passed through the filters 14 r, 14 g, and 14 b of the structured pupil 14.

  The first image sensor 16 is formed by a CCD (Charge Coupled Device) sensor or a CMOS (Complementary Metal-oxide Semiconductor) sensor, and corresponds to a color (in this embodiment, a light beam transmitted through each filter 14r, 14g, 14b. Red, green, and blue) image signals are generated, and the generated image signals of the respective colors are output to the focal position detection unit 17.

  It should be noted that the observation optical system of the microscope and the AF optical system of the autofocus device are all in the same optical arrangement except that the structured pupil 14 is provided. That is, unlike the current active autofocus device, it is not necessary to separately design and incorporate an AF optical system in consideration of the magnification, aberration characteristics, stray light, and the like of the objective lens system 11. Therefore, the apparatus does not become complicated and large, and the manufacturing cost is suppressed.

  In the present embodiment having such a configuration, the light emitted from the specimen S passes through the objective lens system 11 and the pupil magnifying lens system 12, is reflected by the beam splitter 13, and the structured pupil 14 and the first pupil are reflected. The image is formed on the first image sensor 16 through the image forming lens 15, while passing through the beam splitter 13 and imaged on the second image sensor 32 by the second image forming lens system 31. The

  Then, the autofocus device processes and analyzes the image data acquired from the first image sensor 16, detects the focal position of the objective lens system 11, a stage 18 on which the sample S is placed, Focusing control for the specimen S by driving the drive unit 19 based on the detection result by the focus position detection unit 17 and the drive unit 19 that moves at least one of the objective lens system 11 up and down along the optical axis of the objective lens system 11. And a focusing control unit 20 for performing

  For example, a piezo actuator or a stepping motor is used as the drive unit 19. In the present embodiment, the relative position with respect to the specimen S is changed by moving the objective lens system 11 in the optical axis direction by the drive unit 19. However, the stage 18 is moved in the optical axis direction, or the objective lens system 11 and the stage are moved. Both of them may be moved in the optical axis direction.

  Here, the function of the focal position detection unit 17 will be described in more detail. The focal position detection unit 17 transmits an image based on a light beam that is transmitted from the first image sensor 16 through the filters 14r, 14g, and 14b of the structured pupil 14 and separated into a plurality of different color components (red, green, and blue). And the focal position of the objective lens system 11 is detected from the overlapping state of the images based on the light fluxes of the plurality of different color components. This utilizes the principle of color misregistration described below.

For example, in incoherent imaging, or in partial coherent imaging that is almost as close as possible, the pupil plays a role in selecting which rays of the group of rays emitted from the specimen contribute to the imaging. If there is no focus shift, all rays through the pupil will reach the same sensor position. In this case, the position of image formation does not change regardless of the position of the light beam passing through the pupil (see FIG. 2A). On the other hand, when a focus shift occurs, the sensor position where the light beam reaches differs for each position of the light beam passing through the pupil. In this case, the imaging position changes depending on which position of the pupil is selected (see FIG. 2B). That is, when the light ray group L _{1 above} the pupil is selected, an image I ₁ is formed below the optical axis, and when the light ray group L ₂ below the pupil is selected, an image I ₂ is formed above the optical axis. (These images are generally referred to as “parallax images”). The shift amount (distance from the optical axis to the imaging position) at this time depends on the magnitude of the focus shift. This is because if the focus shift is large, the blur of the image also increases, which means that the spread of each parallax image increases.

Based on the principle of such color shift, the image of the sample S acquired through the AF optical system corresponds to the focus shift by the action of the color filters 14r, 14g, and 14b arranged eccentrically on the structured pupil 14. Thus, image misregistration (color misregistration) occurs for each color component. For example, as shown in FIG. 3A, in the structured pupil 14, the red filter 14 r is below the pupil, the green filter 14 g is the right side of the pupil, and the blue filter 14 b is viewed from the light incident direction. When arranged on the right side of the pupil, (1) if in focus, the sample images R ₁ , G ₁ , and B ₁ based on the three color components, red, green, and blue, respectively, Since it is formed at the same position on the image pickup surface of the image pickup device 16, no color shift occurs (see FIG. 3B). (2) If the focal position is shifted forward, the sample image based on the luminous flux of the red component R ₂ is observed on the upper side, the sample image G ₂ based on the light flux of the green component is observed on the right side, and the image B ₂ based on the blue component is observed on the left side (see FIG. 3C). If it is shifted to the side, the color shift direction is reversed. The size of the color shift depends on the size of the focus shift.

  The principle of such color misregistration can be explained using a pupil function and a point spread function (PSF) by optical imaging theory. Hereinafter, for ease of explanation, it is assumed that the AF optical system is an incoherent imaging system and is an aberration optical system at the time of focusing.

  According to the optical imaging theory, when the transfer function of the AF optical system is PSF (point spread function), the object O (x, y), the image I (x, y), and the PSF (x, y) are three. The relationship between the persons is expressed by the following equation (1).

  PSF (x, y), which means a transfer function of the AF optical system, has a point spread amplitude function as ASF (x, y), and the direction cosine of the light beam emitted from the pupil is (ξ, η), the pupil function is G (ξ, η), the pupil amplitude (aperture) is A (ξ, η), and the wavefront aberration representing the aberration of the AF optical system is W (ξ, η). Can be described by the following equation (2).

  The pupil amplitude A (ξ, η) is expressed by the following equation (3) when the numerical aperture of the pupil is a (<1).

  First, let us consider a case where there is no focus shift in the AF optical system. In the present embodiment, since the AF optical system is handled as an aberration-free optical system, the wavefront aberration is W (ξ, η) = 0. Therefore, the point image amplitude distribution ASF (x, y) can be expressed by the following equation (4).

However, C is a constant.

From this state, when the opening position of the pupil is shifted by the numerical aperture s in the ξ and η directions, a new point spread amplitude distribution ASF _shift (x, y) can be expressed by the following equation (5).

Therefore, the point image intensity distribution PSF _shift (x, y) at this time can be expressed by the following equation (6).

  That is, it can be seen that even if the aperture position is shifted in the structured pupil 14, the transfer function PSF of the AF optical system is unchanged, so that it does not affect image formation.

Next, let us consider a case where a focus shift occurs in a state where the pupil aperture position is shifted by the numerical aperture s in the ξ and η directions. The defocus is optically equivalent to adding a second-order wavefront aberration W (ξ, η) = β (ξ ² + η ² ) to the pupil. Here, β is a coefficient indicating the magnitude of the focus shift. Then, the point image amplitude distribution ASF _shift (x, y) can be expressed by the following equation (7).

Here, assuming that the numerical aperture a of the pupil is sufficiently smaller than 1, assuming that the secondary terms ξ ² and η ^{2 are} ignored, the equation (7) can be expressed as the following equation (8).

Therefore, the point image intensity distribution PSF _shift (x, y) at this time can be expressed by the following equation (9).

  This means that the transfer function PSF of the AF optical system is shifted by −2βs while the intensity distribution remains unchanged. Therefore, according to the imaging equation, the image formed through the structured pupil 14 is not changed, and only the imaging position is shifted by −2βs. It can also be seen that the image shift amount depends on the focus shift amount β and the pupil opening position shift amount s.

  Based on the above principle of color misregistration, it can be seen that the sample image based on the luminous flux of each color component separated through the structured pupil 14 causes color misregistration depending on the focus misregistration amount.

Note that the second-order terms ξ ² and η ² ignored in the calculation process have the effect of widening the width of the point image intensity distribution PSF (that is, blur). Therefore, when the numerical aperture a of the pupil is large and these terms cannot be ignored, blurring occurs as the image shifts. This causes a reduction in detection accuracy in the process of calculating a focus shift, which will be described later, so care must be taken in setting the numerical aperture a of the pupil.

Next, a calculation method of the focus shift amount and the shift direction by the focus position detection unit 17 will be described with reference to FIGS. 4 to 6. As shown in FIG. 4, the centers of images R ₃ , G ₃ , and B ₃ based on the light fluxes of the respective color components of red, green, and blue acquired from the first image sensor 16 (in other words, the image formation of the light fluxes of the respective color components). (Position) is I _r , I _g , and I _b , respectively. Then, a virtual central viewpoint is taken as a reference coordinate. When the amount of color misregistration (parallax) at the reference coordinates (x, y) is d pixels, the R (red), G (green), and B (blue) components are I _r (x, y−d) and I _{g, respectively.} It can be expressed as (x + d, y), I _b (x−d, y). Next, a local window w (x, y) is considered around the reference coordinates (x, y), and a set of pixel colors included therein is expressed by the following equation (10).

The positional relationship of this color set is calculated by the following equation (11). However, λ ₀ , λ ₁ , λ ₂ (λ ₀ ≧ λ ₁ ≧ λ ₂ ≧ 0) is a variance along the principal component axis of the color set S (x, y; d) (that is, S (x, y; d) eigenvalues of the covariance matrix Σ), and σ _r ² , σ _g ² , and σ _b ² are along the R (red) axis, G (green) axis, and B (blue) axis of the color set, respectively. Distributed.

The greater the difference between the eigenvalues, the smaller the expression (11), that is, the value of L (x, y; d). If the color set has a linear distribution (λ ₀ >> λ ₁ , λ ₂ ), L is close to 0 and takes the maximum value L = 1 when completely uncorrelated. That is, as shown in FIG. 5, when there is no color misalignment, L = 0 (In-focus), and as the color misregistration increases, the value of L increases (Defocus-1, Defocus-2). However, since L always takes a positive value, the direction of focus shift of the objective lens system 11 cannot be determined. Therefore, the following method shown in FIG. 6 is taken.

First, images R ₄ , G ₄ , and B ₄ based on the luminous fluxes of the red, green, and blue color components in the acquired local window w (x, y) are displayed in the direction of color shift (R axis, G axis, Images shifted by ± p pixels (along the B axis) are prepared in the computer, and the values of L corresponding to these are calculated as L ₊ and L ₋ . When the focus shift is on the rear side, the images R ₄ , G ₄ , and B ₄ approach each other when shifted by −p pixels, and the images R ₄ , G ₄ , and B ₄ move toward each other when shifted by + p pixels. away, in other words L _+> L> L _- holds relationship. When the focus shift is on the front side, the R ₄ , G ₄ , and B ₄ images move away from each other when shifted by −p pixels, and the R ₄ , G ₄ , and B ₄ images approach each other when shifted by + p pixels. That is, the relationship L ₋ >L> L ₊ holds. In this way, in addition to calculating the value of L and confirming the presence or absence of color misregistration, calculating the values of L ₊ and L ₋ and confirming the magnitude relationship at the same time, the direction of focus misalignment (front side) Or the rear side).

  Note that, as the value of p, one pixel, which is the minimum amount of parallax, is considered appropriate, but it is not limited to a specific value. In addition, since the calculation of L gives the structure of the sample image, if the image is greatly blurred and the structure becomes unclear, there is a high possibility that the measurement accuracy will also decrease at the same time. Therefore, pay sufficient attention to the opening size of the structured pupil 14.

  Subsequently, the measurement resolution (depth resolution) Δz of the focus shift calculated by the focus position detection unit 17 will be described. The focus shift measurement resolution Δz can be expressed by the following equation (12) when estimated by paraxial approximate geometric optics assuming a thin lens.

Here, D is the relative distance between the pupil center positions of the filters 14r, 14g, and 14b of the structured pupil 14, f _obj is the focal length of the objective lens system 11, F _obj is the F number of the objective lens system 11, and γ is The magnification of the external extension pupil by the pupil magnifying lens system 12, f ₃ is the focal length of the first imaging lens system 15, and ps is the pixel size of the first image sensor 16.

That is, when it is desired to improve the resolution, the relative distance interval D between the pupil center positions of each filter of the structured pupil 14 is increased, or the pixel size ps, the focal length f _{obj of} the objective lens system 11, and the objective lens system 11 What is necessary is just to design so that F number _Fobj etc. may be made small.

For reference, a resolution of Δz = 0.3 μm can be achieved under the conditions of f _obj = 20 mm, F _obj = 1.5, γ = 5, f ₃ = 200 mm, ps = 4.5 μm, and D = 30 mm.

  FIG. 7 shows a series of processes from the measurement of the focus shift by the focus position detection unit 17 to the correction of the focus position by relatively moving the objective lens system 11 and the sample S by the focus control unit 20 in the autofocus device. Show the process. Here, the same real-time feedback is performed as in the current AF.

  First, the entire image is divided into a plurality of windows, and L is calculated for each window. Among these, the window in which the value of L takes the minimum value is determined as the initial focus position. In the following processing, only this window portion is set as an L calculation target. The focal position detection unit 17 acquires R (red) component, G (green) component, and B (blue) component images for each frame from the first image sensor 16 (step ST1).

Next, the focal position detection unit 17 calculates the values of L ₋ , L, and L ₊ based on the above-described color deviation amount calculation method (see FIG. 6), and determines the focal deviation amount and the deviation direction (see FIG. 6). Step ST2).

  Note that in step ST2, the image capture and the shift amount calculation are directly executed by hardware (not external processing) provided in the autofocus device. Although a method of taking a captured image once in a personal computer and calculating it in the personal computer is also conceivable, it is not preferable from the viewpoint of processing and communication speed.

  Then, the focal position detection unit 17 determines whether or not the value of L is a target value of zero (step ST3). Here, when the value of L is zero, that is, in the in-focus state, the process returns to step ST1. On the other hand, if the value of L is not zero, that is, if a focus shift has occurred, the process proceeds to step ST4.

  The focus control unit 20 derives a correction value (additional voltage value to the drive unit 19) from the focus shift amount and the shift direction detected by the focus position detection unit 17 (step ST4).

  In this embodiment, the objective lens system 11 is finely moved in the optical axis direction by the drive unit 19 to correct the focus shift. The relationship between the amount of additional voltage applied to the drive unit 19 and the amount of movement of the objective lens system 11 is calibrated in advance. Based on this relationship, the focus control unit 20 converts the focus shift amount into a correction value (additional voltage value to the drive unit 19).

  Finally, the drive unit 19 is driven based on the correction value (additional voltage value) to finely move the objective lens system 11 to correct the focus shift (step ST5).

  Real-time autofocus control is realized by always performing such a feedback process.

  In the present embodiment, since processes such as “image acquisition”, “focus shift amount calculation”, and “correction value conversion” are performed, the actual autofocus (AF) response speed is determined by the focus shift amount by hardware. That is, the calculation processing speed and the maximum frame rate of the sensor allowed in the range (speed at which a series of images can be acquired). Here, as an estimation of the calculation speed by hardware, referring to the research field of real-time stereo camera development that performs almost the same calculation processing using an FPGA device, an image with a resolution of 640 × 480 pixels and a frame rate of about 270 fps is obtained. Real-time processing at high speed is possible. Therefore, as the AF response speed in this embodiment, a high speed of the order of several tens of ms can be expected, which can be said to be sufficient even when compared with the current AF response speed.

  As another form for realizing the same effect as that of the first embodiment (see FIG. 1), a form in which the object can be realized by the same optical system without dividing the observation optical system and the AF optical system is also considered. It is done. FIG. 8A shows a configuration diagram of the optical system. In this embodiment, there is no AF optical system, a transmissive liquid crystal display element LCD (Liquid Crystal Display) is disposed immediately above the structured pupil, and the shape of the structured pupil is changed. This is different from the configuration shown in FIG. Hereinafter, the same members as those in the embodiment of FIG. 1 are denoted by the same reference numerals, and description thereof is omitted.

  In the structured pupil 14 ', the plate-like member 14a' has a donut shape, a plurality of openings 14h 'are formed in the ring portion, and files 14r, 14b, and 14g are attached to the respective openings 14h'.

  Two transmissive mask patterns are added to the transmissive liquid crystal display element LCD. Here, a first mask pattern having a circular opening 21 (see the left figure in FIG. 8B) and a second mask pattern having a ring-shaped opening 22 shown in FIG. 8 (c), see the left figure).

  Note that the size of the opening 21 of the first mask pattern is substantially equal to the size of the opening of the structured pupil 14 '. Therefore, when the structured pupil 14 ′ and the first mask pattern are combined, the objective lens system via the pupil magnifying lens system 12 is provided on the imaging surface of the second imaging element 32 by the second imaging lens system 31. 11 is directly imaged. Further, the width of the opening 22 of the second mask pattern is substantially equal to the width of the ring portion of the structured pupil 14 '. Therefore, when the structured pupil 14 ′ and the second mask pattern are combined, the second imaging lens system 31 passes through each filter of the structured pupil 14 to the imaging surface of the second imaging element 32. Only the luminous flux of the color component is imaged.

  Therefore, when the specimen S is photographed, the first mask pattern shown in FIG. 8B is added to the liquid crystal display element LCD. As a result, the configuration is the same as that of the observation optical system in FIG. 1, and a pure image of the specimen S can be acquired (normal mode). On the other hand, when detecting the focus shift, the second mask pattern shown in FIG. 8C is added to the liquid crystal display element LCD. As a result, the configuration is the same as that of the AF optical system in FIG. 1, and information regarding the focus shift can be acquired (AF mode).

  The switching of the mask pattern of the liquid crystal display element LCD and the timing of image acquisition by the second image sensor 32 are synchronized. That is, the normal mode and the AF mode of the liquid crystal display element LCD are alternately switched in accordance with the frame rate of the second image sensor 32. That is, the sample image and the focus shift information are obtained alternately.

  This method has disadvantages such as halving the frame rate of the image of the specimen S, reducing the optical resolving power, and causing a diffraction phenomenon due to the pitch structure of the liquid crystal display element LCD, but it is necessary to prepare an AF optical system separately. There is no merit in that there is a merit that the size and cost can be reduced. Further, when the diffraction phenomenon becomes a problem, as shown in FIG. 9, it can be solved by separately creating and using a liquid crystal display element having a donut shape (modulation portion is a ring type) instead of a conventional pixel structure. .

  The advantages of the first embodiment are summarized here. (1) It is not necessary to separately design and incorporate an AF optical system that is independent from the observation optical system. (2) Focus deviation is a color component. Is read from the overlapping state of different specimen images, so even if the alignment of the optical system fluctuates for some reason, it has little effect on detection accuracy, and stable focus detection can be performed. (3) Extremely simple processing and high speed A simple autofocus device can be realized.

(Second Embodiment)
FIG. 10 shows a configuration example of a microscope in which the autofocus device according to the second embodiment is incorporated. In FIG. 10, only the optical system for AF is shown for simplicity of explanation, and the optical system for observation for photographing the specimen S is omitted. Hereinafter, the same members as those in the first embodiment are denoted by the same reference numerals, and description thereof is omitted.

  In the second embodiment, an active method is adopted as the autofocus device. In this method, an optical pattern for AF is projected onto the sample surface, and the reflected light is used as an autofocus index. This method is advantageous in that it can detect a focus shift of the entire field of view, including information on the tilt (hereinafter referred to as tilt) of the specimen surface.

  The autofocus device according to the second embodiment includes a Koehler illumination system 40, an objective lens system 11, a beam splitter 13, a pupil magnifying lens system 12, a structured pupil 14, and a structured pupil 14 arranged in order along the optical axis from the sample side. An AF optical system including the first imaging lens system 15 and a first image sensor 16 are included.

  The Kohler illumination system 40 includes an AF light source (white light source) 41 that emits light having a wavelength width, a condenser lens 41, and a projection mask in which a pattern having a light transmitting region is repeatedly formed along a predetermined direction. 43 and a relay lens system 44 (including the objective lens system 11 and the beam splitter 13). The projection mask 43 may be provided with a fixed opening in advance, or may be electrically generated by a spatial light modulator or the like.

  In the second embodiment, first, an image of the pattern of the projection mask 43 illuminated by the light emitted from the AF light source 41 via the condenser lens 42 is projected onto the specimen S via the relay lens system 44. Here, for example, a projection mask 43 having a pattern in which a round structure is projected uniformly over the entire field of view as shown in FIG. 10 is assumed.

The pattern image reflected by the sample surface is reflected by the beam splitter 13 and travels to the AF optical system. The subsequent optical configuration and the shape of the structured pupil conform to the first embodiment. The image captured by the first image sensor 16 causes a color shift corresponding to the focus shift amount of the objective lens system 11. However, unlike the first embodiment, as shown in FIG. 11, the color at each formation position on the imaging surface is different from the pattern image _{S 1, S 2, ··· S} n-1, S n If the amount of deviation is detected, it is possible to obtain focus deviation information for the entire visual field area including information on the inclination of the sample surface.

  In the present embodiment, the method of calculating the focus shift amount and the shift direction and the process of feedback control are the same as those in the first embodiment described above. However, the following calculation processing is added in order to estimate the tilt information of the sample surface (see FIG. 11).

First, a numerical map (matrix) of the focus shift amount L is prepared with reference to the position of the pattern projected on the imaging surface. This is assumed to be a plane, and this matrix is rearranged into a one-dimensional vector and denoted as M. Here, X, Y, and Z are prepared by preparing a base plane matrix that expresses an inclination in the x direction, an inclination in the y direction, and a focus shift in the z direction (optical axis direction), and rearranges the matrix into a one-dimensional vector. Is written. In addition, the coefficients related to these are expressed as a _x , a _y and a _z , respectively. By fitting the coefficients a _x , a _y, and a _z with respect to M by the least square method, it is possible to estimate the tilt information of the sample surface. Specifically, it is obtained from the following equation (13).

A ⁺ is a pseudo inverse matrix of A.

Based on the obtained coefficients a _x , a _y, and a _z, they are converted into correction amounts in the respective axial directions, and this is used as a feedback index.

  As a three-dimensional focus shift correction mechanism, for example, as shown in FIG. 12, three piezo stages 52, 53, and 54 are attached to the lower part of the sample stage 51. The plane deviation in the x-axis direction is corrected by moving the piezo stages 52 and 53 simultaneously. The plane deviation in the y-axis direction is corrected by moving the piezo stages 53 and 54 simultaneously. The defocus in the z-axis direction is corrected by moving all the piezo stages 52, 53, and 54 simultaneously.

  Here, the advantages of the second embodiment are summarized as follows: (1) Since the focus shift is read from the overlapping state of the sample images having different color components, even if the alignment of the optical system fluctuates for some reason, the detection accuracy is improved. (2) The focus shift information can be collected by extremely simple and high-speed processing, and the tilt information of the sample surface is added, and the focus shift and The correction can be realized.

  In order to make the present invention easy to understand, the configuration requirements of the embodiment have been described, but it goes without saying that the present invention is not limited to this.

DESCRIPTION OF SYMBOLS 11 Objective lens system 14 Structured pupil 14a Plate-shaped member 14h Structured pupil aperture 14r, 14b, 14g Filter 15 First imaging lens system (AF optical system)
16 First image sensor (AF optical system)
17 focus position detection unit 18 stage 19 drive unit 20 focusing control unit 31 second imaging lens system (observation optical system)
32 Second image sensor (observation optical system)
S specimen

Claims (13)

An objective lens system that collects the luminous flux from the specimen;
On the exit side of the objective lens system, a plate-like member that is provided at an exit pupil position of the objective lens system or a position substantially conjugate with the pupil position, and shields the light beam from the objective lens system; and A structured pupil having a plurality of formed openings and a filter that is attached to the plurality of openings and transmits only a light beam of a predetermined color component;
An imaging lens system that forms an image on the imaging device with a light beam transmitted through each filter of the structured pupil,
Processing and analyzing image data acquired from the image sensor, and having a focal position detection unit that detects a focal position of the objective lens system;
The focal position detection unit acquires an image based on a light beam that has been transmitted from the image sensor through each filter of the structured pupil and separated into a plurality of different color components, and an image based on the light beams of the plurality of different color components. An autofocus device that detects a focal position of the objective lens system from the degree of overlap of the objective lens system.   2. The autofocus device according to claim 1, wherein the plurality of apertures are formed at positions decentered with respect to a center of an exit pupil of the objective lens system.   The focus position detection unit determines that the objective lens system is in a focus position when images based on light beams separated into a plurality of different color components transmitted through the filters of the structured pupil all overlap. The autofocus device according to claim 1 or 2.   The auto filter according to any one of claims 1 to 3, wherein the filter is a filter that transmits only a red light beam, a filter that transmits only a green light beam, and a filter that transmits only a blue light beam. Focus device.   The focal position detection unit determines the presence and direction of a focal shift using a positional relationship of a set of pixel colors included in a local window that is a part of the image acquired by the imaging device. The autofocus device according to any one of claims 1 to 4, wherein   The focus position detection unit determines that there is no focus shift when linearity exists in the set of pixel colors included in the local window, and determines that there is focus shift when linearity does not exist. The autofocus device according to claim 5.   The focal position detection unit calculates a value of L using a predetermined calculation formula where L is an index for measuring the linearity of a set of pixel colors included in the local window. 9. The autofocus device according to claim 8, wherein it is determined that there is a focus shift and there is no focus shift, and if L ≧ α, there is no linearity and it is determined that there is a focus shift (where α is not limited) Value close to 0). The focal position detection unit shifts an image based on the light flux of each color component in the local window by + p pixels with respect to the direction of color shift in the local window, where L is an index for measuring the linearity of a set of pixel colors included in the local window. An index for measuring the linearity of a set of pixel colors is assumed to be L _+, and a pixel when it is assumed that an image based on the luminous flux of each color component is shifted by −p pixels in the color shift direction in the local window. when a, L by using the predetermined formula, L _+, L _{- -} a measure of linearity of the color set of L values to the respectively calculated, L _+> L> L _- when the relationship is established that determines that the later direction of defocus side (the subject side), L _-> L> when L ₊ is the direction of defocus when the relation holds that is the front side (the objective lens system side) It is characterized by judging The autofocus device according to claim 5 or 6. 9. The autofocus device according to claim 7, wherein the predetermined calculation formula is represented by the following formula.
L = λ ₀ λ ₁ λ ₂ / σ _r ² σ _g ² σ _b ²
However,
L: an index for measuring the linearity of a set of pixel colors included in the local window;
λ ₀ , λ ₁ , λ ₂ (λ ₀ ≧ λ ₁ ≧ λ ₂ ≧ 0): dispersion along the principal component axis of a set of pixel colors included in the local window,
σ _r ² , σ _g ² , σ _b ² : dispersion along the R axis, G axis, and B axis of the set of colors. A pupil magnifying lens system for enlarging and relaying the exit pupil of the objective lens system;
10. The structured pupil is provided at a position substantially conjugate with an exit pupil of the objective lens system enlarged and relayed by the pupil projection lens system. The autofocus device described in 1. The projection that has a light source that emits light having a wavelength width and a projection mask in which a pattern having a light transmitting region is repeatedly formed along a predetermined direction, and is illuminated by the light emitted from the light source A Koehler illumination system that projects an image of a mask pattern onto the specimen surface via the objective lens system;
The focal position detection unit obtains, from the image sensor, an image based on a light beam that is transmitted through each filter of the structured pupil and separated into a plurality of different color components for each position of the pattern projected on the specimen surface. The focus position of the objective lens system and the tilt information of the sample surface are detected from the degree of overlap of images based on the light beams of the plurality of different color components. The autofocus device described in 1. A stage on which the specimen is placed;
A drive unit that moves up and down at least one of the stage and the objective lens system along an optical axis of the objective lens system;
The focus control unit that drives the drive unit based on a detection result by the focus position detection unit and changes an interval between the sample and the objective lens system. The autofocus device according to claim 1.   A microscope comprising the autofocus device according to any one of claims 1 to 12.