Microscope system

Abstract

It provides high-speed and high-precision autofocus. [Solution] The splitter 105 splits the light from the sample S supported by the stage 103, which has passed through the objective lens 104, into the optical axis of the observation optical system and the optical axis of the detection optical system. The focus evaluation value calculation unit 164 calculates a focus evaluation value for the image of the sample S at first and second positions on the optical axis of the detection optical system, which are in front of and behind the position optically conjugate to the front focal point of the objective lens 104, based on the contrast of the image of the sample S at each of these positions. The focusing control unit 140 obtains focus evaluation values ​​from the focus evaluation value calculation unit 164 for each of the images of a plurality of samples S whose spatial frequency components constituting the image of the sample S are different from each other, estimates a focus evaluation value for the image of the sample S composed of predetermined spatial frequency components using the obtained focus evaluation values, and controls the focusing unit 150 based on the estimation result.

Description

Technical Field

[0001] The disclosure of this specification relates to a microscope system.

Background Art

[0002] As one of the technologies for reducing the burden on pathologists in pathological diagnosis, WSI has been attracting attention. WSI is an abbreviation for Whole Slide Imaging, which is a technology for scanning the entire specimen on a slide glass to create a digital image, and by acquiring and tiling a plurality of images, it is a technology for imaging a wider area than the field of view of a microscope with high resolution. In order to improve the throughput of this WSI, high-speed and high-precision autofocus (hereinafter, referred to as "AF") is required.

[0003] As a technology related to WSI, a technology that achieves both high image quality and high throughput at a high level is known (see, for example, Patent Document 1).

[0004] Also, as a technology related to AF, a technology is known in which a clear microscopic image of a sample is obtained in a short time by focusing on a wide range of a sample having unevenness on its surface or the structure inside the sample (see, for example, Patent Document 2).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] One type of autofocus (AF) known for its high processing speed is the optical path difference method. The optical path difference AF method controls the placement of a sample so that the contrast values ​​of the sample image detected at positions in front of and behind the focal point of the imaging optical system match.

[0007] In optical path length difference autofocus (AF) systems, various structures exist, and focusing accuracy can decrease when dealing with thick tissue specimens. This decrease in accuracy can be caused by spherical aberration resulting from the cover glass protecting the specimen on the slide, and differences in pixel size between the image sensor used for acquiring microscope images and the light detection sensor used for AF.

[0008] In view of the above points, one aspect of the present invention is to provide high-speed and high-precision autofocus. [Means for solving the problem]

[0009] A microscope system according to one aspect of the present invention comprises a stage, an objective lens, a focusing unit, a branching unit, a sensor, a focus evaluation value calculation unit, an image sensor, and a control unit. The focusing unit changes the distance between the stage and the objective lens. The branching unit branches the light from the sample supported by the stage, which has passed through the objective lens, into the optical axis of the observation optical system and the optical axis of the detection optical system. The sensor acquires brightness values ​​for the image of the sample at a first position and a second position on the optical axis of the detection optical system. The first position is a position in front of the position optically conjugate to the front focal point of the objective lens, and the second position is a position behind the optically conjugate position. The focus evaluation value calculation unit calculates a focus evaluation value for the image of the sample based on the contrast obtained from the brightness values ​​at the first position and the second position acquired by the sensor. The image sensor captures an image of the sample formed on the optical axis of the observation optical system. The control unit obtains focus evaluation values ​​from the focus evaluation value calculation unit for each of several images of the sample, each of which has different spatial frequency components constituting the sample image. The control unit also uses the obtained focus evaluation values ​​to estimate the focus evaluation value for the sample image composed of predetermined spatial frequency components, and controls the focusing unit based on the estimation result. [Effects of the Invention]

[0010] According to the above embodiment, high-speed and high-precision autofocus can be provided. [Brief explanation of the drawing]

[0011] [Figure 1] This diagram shows an example of the configuration of a microscope system. [Figure 2] This is a diagram explaining WSI. [Figure 3] This is a diagram showing an example of a sensor structure. [Figure 4] This diagram explains the method for calculating the focus evaluation value. [Figure 5] This diagram illustrates the positional shift in the detection result of the focus position in autofocus using the optical path length difference method. [Figure 6]It is a diagram for explaining the cause of the positional deviation of the detection result of the in-focus position in AF by the optical path length difference method. [Figure 7] It is a diagram for explaining a method of estimating the focus evaluation value. [Figure 8] It is a flowchart showing an example of the procedure of the AF control method. [Figure 9] It is a diagram showing a first example of an approximate curve obtained by function fitting. [Figure 10] It is a diagram for explaining a second example of the estimation method. [Figure 11] It is a diagram showing a second example of an approximate curve obtained by function fitting. [Figure 12] It is a diagram for explaining a first example of a method for selecting a target for calculating the focus evaluation value. [Figure 13] It is a diagram for explaining a second example of a method for selecting a target for calculating the focus evaluation value. [Figure 14] It is a diagram showing a first example of an approximate curve obtained by function fitting for a weighted value. [Figure 15] It is a diagram showing a second example of an approximate curve obtained by function fitting for a weighted value.

Mode for Carrying Out the Invention

[0012] Hereinafter, embodiments will be described in detail while referring to the drawings.

[0013] FIG. 1 is a diagram showing an example of the configuration of the microscope system 1. FIG. 2 is a diagram for explaining WSI. Hereinafter, the configuration of the microscope system 1 will be described while referring to FIGS. 1 and 2.

[0014] The microscope system 1 is a system used, for example, for pathological diagnosis applications and has the function of WSI.

[0015] As shown in Figure 1, the microscope system 1 comprises a microscope device 100 and an image processing device 200. The microscope device 100 uses an objective lens 104 to scan a specimen wider than the actual field of view and outputs multiple images of the sample S to the image processing device 200.

[0016] When the microscope apparatus 100 images the entire sample S sandwiched between a glass slide SG and a cover glass CG, as shown in Figure 2, using WSI, it scans the entire sample S by repeatedly taking images while moving the real field of view 10 relative to the sample S. At this time, the imaging position is set so that the image of the sample S obtained in each imaging is overlapped with a portion of the image taken at an adjacent imaging position (see the stitching area in Figure 2).

[0017] The image processing device 200 synthesizes multiple images of the sample S output from the microscope device 100 to generate a stitched image. Specifically, the image processing device 200 generates a WSI image by stitching together multiple images using the overlapping portions of images captured at adjacent imaging positions as stitching margins. This allows for accurate identification of the relative positions of multiple images and stitching them together.

[0018] As shown in Figure 1, the microscope apparatus 100 comprises at least a light source 101, an objective lens 104, a stage 103, a two-dimensional image sensor 170, a focusing unit 150, and a control unit 180. The control unit 180 may include one or more control circuits. The control unit 180 may also include a microscope control unit 110, a light source control unit 120, a stage control unit 130, and a focusing control unit 140, and each of these may further include one or more control circuits (control circuit 111, control circuit 121, control circuit 131, control circuit 141).

[0019] The microscope apparatus 100 includes a transmitted illumination optical system that illuminates the sample S supported on the stage 103, on the opposite side of the objective lens 104 across the stage 103. More specifically, the microscope apparatus 100 includes a light source 101 and a condenser lens 102 as the transmitted illumination optical system. The light source 101 is, for example, a light-emitting diode, but is not particularly limited to a light-emitting diode. The light source 101 is configured to emit light in accordance with the input from the light source control unit 120.

[0020] The objective lens 104 moves in the optical axis direction by the operation of the focusing unit 150. The objective lens 104 has, for example, a numerical aperture (NA) of 0.4 and a magnification of 40x. However, the objective lens 104 only needs to have an NA that can achieve the resolution required by the user for the stitched image, and the magnification of the objective lens 104 is not limited to 40x.

[0021] For example, in pathological diagnosis, the cells used as sample S have a diameter of approximately 10 μm (micrometers). Therefore, in order to observe the aggregation of cell nuclei using the microscope system 1, it is preferable that the microscope system 1 provides a resolution higher than approximately 1.0 μm. For example, if the observation wavelength λ in visual observation is 550 nm (nanometers) and the NA of the objective lens 104 is 0.3, and Rayleigh's resolution formula is applied, it can be seen that a resolution δ of the above degree can be obtained (see formula [Equation 1]).

[0022]

number

[0023] Therefore, for pathological diagnostic applications, it is preferable that the numerical aperture (NA) of the objective lens 104 be 0.3 or higher.

[0024] Stage 103 includes an XY stage that moves in the XY direction perpendicular to at least the optical axis of the objective lens 104. Stage 103 may further include a Z stage that moves in the direction of the optical axis. Stage 103 is configured to move according to input from the stage control unit 130. Stage 103 may include actuators, for example, a stepping motor and a ball screw (not shown), and the position of stage 103 may be controlled, for example, by controlling the actuators in an open-loop manner.

[0025] The two-dimensional image sensor 170 is, for example, a CMOS image sensor and captures an image of the sample S that is mounted and supported on the stage 103. The two-dimensional image sensor 170 has an electronic shutter, such as a rolling shutter or a global shutter. The two-dimensional image sensor 170 is configured so that at least the timing of exposure start is controlled according to a control signal from the microscope control unit 110.

[0026] More specifically, in the two-dimensional image sensor 170, the timing of the exposure start and end may be controlled, for example, by the pulse width of the control signal. Alternatively, in the two-dimensional image sensor 170, exposure may end after a preset time has elapsed from the exposure start timing specified by the control signal. In other words, the microscope control unit 110 is configured to perform exposure control to control the exposure period of the two-dimensional image sensor 170.

[0027] The focusing unit 150 changes the distance between the stage 103 and the objective lens 104. In this embodiment, the focusing unit 150 is configured to move the objective lens 104 in the optical axis direction according to the input from the focusing control unit 140. The focusing control unit 140 is configured to perform focusing control, for example, by controlling the focusing unit 150 at a predetermined sampling period so that the focus is brought into focus on the sample S, when instructed by the microscope control unit 110 to perform real-time AF. More specifically, the focusing control unit 140 controls the focusing unit 150 based on the focus evaluation value output from the focusing unit 160, which will be described later.

[0028] In the microscope system 1 configured as described above, the microscope device 100 scans the target area by capturing an image of the sample S without stopping the stage 103 at the imaging position. In other words, during the movement period when the objective lens 104 is moving in a direction perpendicular to the optical axis of the stage 103, the control circuit included in the control unit 180 performs exposure control to control the exposure period of the two-dimensional image sensor 170.

[0029] Furthermore, in the microscope system 1, the microscope device 100 performs autofocus in real time during scanning. That is, during the movement period when the objective lens 104 is moving in a direction perpendicular to the optical axis of the stage 103, the control circuit included in the control unit 180 performs focus control by controlling the focusing unit 150 based on the focus evaluation value detected during the movement period. More specifically, the focus evaluation period during which the focus evaluation value is detected is set to a predetermined sampling period, so that the control circuit performs focus control at a predetermined sampling period. This suppresses focus shifts at each imaging position, making it possible to generate high-quality images using WSI.

[0030] Therefore, the microscope system 1 makes it possible to achieve a high level of both image quality and throughput, and high-quality images can be obtained in a short time using WSI.

[0031] The focusing unit 160 outputs the differential contrast, which is the difference in contrast detected by the light detection sensor, as the focus evaluation value. The focusing unit 160 is located on the AF optical path that is branched off from the optical path between the objective lens 104 and the 2D image sensor 170 by the splitter 105.

[0032] The splitter 105 splits the light from the sample S supported by the stage 103, which has passed through the objective lens 104, into the optical axis of the observation optical system that goes towards the two-dimensional image sensor 170 via the imaging lens 106, and the optical axis of the detection optical system that goes towards the focusing unit 160. In other words, the splitter 105 is an example of a branching section that splits the light from the sample S supported by the stage 103, which has passed through the objective lens 104, into the optical axis of the observation optical system and the optical axis of the detection optical system. The splitter 105 only needs to guide at least a portion of the incident light into the optical path for AF that goes towards the sensor 163, and may be, for example, a half mirror.

[0033] The focusing unit 160 includes a condensing lens 161, a splitter 162, a sensor 163, and a focus evaluation value calculation unit 164.

[0034] The focusing lens 161 is a lens equivalent to the imaging lens 106, positioned between the objective lens 104 and the two-dimensional image sensor 170, and forms an image of the sample S on the optical axis of the detection optical system. The imaging lens 106 forms an image of the sample S on the optical axis of the observation optical system, and the two-dimensional image sensor 170 captures the image of the sample S formed by the imaging lens 106 on the optical axis of the observation optical system.

[0035] The splitter 162 is a half-mirror positioned between the condensing lens 161 and the sensor 163, and it splits the light that has passed through the condensing lens 161 into two.

[0036] Sensor 163 will be explained with reference to Figure 3. Figure 3 shows an example of the structure of sensor 163.

[0037] Sensor 163 is an example of a photodetector, such as an array sensor with multiple pixels arranged in a row. The two beams of light split by the splitter 162 travel from the sample S through different optical path lengths and enter different regions of sensor 163. More specifically, one of the two different regions of sensor 163 detects one beam of light in front of the position optically conjugate to the front focal point of the objective lens 104 (i.e., the focus position), and the other of the two different regions of sensor 163 detects the other beam of light behind the said focus position. That is, sensor 163 detects light at a first position in front of the position optically conjugate to the front focal point of the objective lens 104 (front focus position) and a second position behind it (rear focus position).

[0038] The sensor 163 is not particularly limited, but for example, it may be a line sensor with multiple pixels aligned in a certain direction, as shown in Figure 3. When the sensor 163 is a line sensor, the multiple pixels of the sensor 163 are arranged to align in the direction (y direction) corresponding to the main scanning direction (Y direction). By arranging the sensor 163 in this way, as shown in Figure 3, it becomes possible to detect light with different optical path lengths by the distance between two regions (first region 163a and second region 163b) that are spaced apart in the y direction of the sensor 163. In other words, the y direction can also be used as the direction (z direction) corresponding to the optical axis direction (Z direction) on the sample S. As a result, as shown in Figure 7, the sensor 163 having the first region 163a and the second region 163b alone can detect light at the first position (front focus position) and the second position (rear focus position) as described above. Of course, the sensor 163 may also consist of a first sensor having a first region 163a for detecting light at a first position (front focus position) and a second sensor having a second region 163b for detecting light at a second position (rear focus position).

[0039] In the following explanation, "front focus position" will be abbreviated as "front pin," and "rear focus position" will be abbreviated as "rear pin."

[0040] Next, the focus evaluation value calculation unit 164 will be described. The focus evaluation value calculation unit 164 calculates a focus evaluation value for detecting the state of focus. The method for calculating this focus evaluation value will be explained with reference to Figure 4.

[0041] As shown in Figure 4, the focus evaluation value calculation unit 164 first acquires the luminance value of light from the sample S detected by each pixel in the sensors 163 (first region 163a and second region 163b) which are located in the front and rear focus areas, respectively. Then, in order to bin a predetermined number (for example, 4 pixels) of adjacent pixels into a single pixel, the acquired luminance values ​​are summed up in the order of the pixel positions, and the resulting sum is taken as the luminance value for the binned single pixel.

[0042] Next, the focus evaluation value calculation unit 164 uses the obtained luminance values ​​to calculate the contrast of the image of the sample S at front focus and back focus, respectively, according to the calculation formula shown in Figure 4. That is, the focus evaluation value calculation unit 164 calculates the contrast value C as the sum of the squares of the differences in luminance values ​​of two adjacent pixels.

[0043] Next, the focus evaluation value calculation unit 164 obtains a focus evaluation value E for the image of the sample S by subtracting the contrast value Cf from the contrast value Cb, based on the contrast value Cf at the front focus and the contrast value Cb at the back focus. This focus evaluation value E is sometimes called the differential contrast. The focus evaluation value calculation unit 164 outputs the focus evaluation value E obtained in this way to the control unit 180.

[0044] The focus evaluation value E is 0 (zero), which indicates focus when the sample S is located at the focus position of the objective lens 104.

[0045] Furthermore, the focal evaluation value E is negative when the sample S is located closer to the objective lens 104 than the focal point of the objective lens 104. Conversely, the focal evaluation value E is positive when the sample S is located further away from the objective lens 104 than the focal point of the objective lens 104. In this way, the sign (positive or negative) of the focal evaluation value E allows us to identify the direction in which the focal point exists.

[0046] The focusing control unit 140 in the control unit 180 controls the focusing unit 150 using the optical path length difference method based on this focus evaluation value E. More specifically, the focusing control unit 140 determines the direction in which to move the objective lens 104 using the sign of the difference contrast, and further determines the amount and speed of movement of the objective lens 104 using the absolute value of the difference contrast. By controlling the focusing unit 150 according to these determinations, high-speed focusing can be achieved in the microscope system 1.

[0047] As described above, the microscope system 1 employs an optical path difference type of autofocus (AF) which is suitable for use in real-time AF during movement. The optical path difference type of AF can determine the direction in which the position (z position) of the objective lens 104 should be moved by understanding the direction in which the focus position exists, and thus can quickly focus. However, with the optical path difference type of AF, the detection result of the focus position in the detection optical system may deviate from the focus position in the observation optical system, which can reduce the accuracy of the focus. The positional deviation of the detection result of the focus position in the optical path difference type of AF will be explained with reference to Figure 5.

[0048] The horizontal axis of the three graphs shown in Figure 5 all represents the z position of the objective lens 104.

[0049] The two curves labeled [A] in Figure 5 represent an example of the relationship between the z position of the objective lens 104 and the contrast of the image of the sample S at the front and rear focus positions, respectively. In the following explanation, such a curve representing the relationship between the z position of the objective lens 104 and the contrast of the image of the sample S will be referred to as a "contrast curve".

[0050] Furthermore, the graph [B] in Figure 5 is a curve that shows the relationship between the z position of the objective lens 104 and the focal evaluation value, calculated from the data represented by the two contrast curves in [A]. In this graph [B], the position where the focal evaluation value E is 0 (zero), that is, the z position that intersects the horizontal axis, is the determined focus position, or in other words, the detection result of the focus position using the optical path length difference method.

[0051] On the other hand, graph [C] in Figure 5 is an example of a contrast curve for sample S obtained by the two-dimensional image sensor 170. The z position where the contrast of the image of sample S obtained by the two-dimensional image sensor 170 is maximum can be said to be a highly accurate focus position. This type of AF method, which uses the z position where the contrast of the image of sample S is maximum as the focus position detection result, is widely known as the contrast method.

[0052] In Figure 5 [C], the z position where the contrast of the image of sample S is maximized is referred to as the "accurate focus position." However, in the example in Figure 5, there is a positional shift between the accurate focus position shown in the graph of [C] and the judged focus position shown in the graphs of [A] and [B]. The cause of this positional shift will be explained with reference to Figure 6.

[0053] Sample S is a thick tissue specimen with various structures, and such a tissue specimen is placed on a glass slide SG as sample S and protected by a cover slip CG. Therefore, when observing such a sample S, spherical aberration occurs due to differences in the thickness of the cover slip CG and the tissue section, and differences in the refractive index of the mounting material and the tissue, causing the degree of image blurring described above to change. This spherical aberration changes the z position of the objective lens 104 where the contrast is maximum in the contrast curve.

[0054] The graph in Figure 6 shows examples of contrast curves for the low and high spatial frequency components of the image of sample S. Of these, contrast curve [A] represents the ideal state unaffected by spherical aberration as described above, while contrast curve [B] represents the actual behavior affected by spherical aberration as described above. Comparing the contrast curves [A] and [B], it can be seen that in this example, the contrast curve for the low spatial frequency component is more significantly affected by spherical aberration. Thus, the degree of influence of spherical aberration on the contrast curve of the spatial frequency components of an image differs depending on the spatial frequency.

[0055] In the observation optical system, the two-dimensional image sensor 170 that captures an image of the sample S uses small pixels to obtain an image of its fine structure. Therefore, the acquired image can sufficiently contain spatial frequency components from low to high spatial frequencies. On the other hand, the sensor 163 that captures an image of the sample S in the detection optical system has larger pixels compared to the two-dimensional image sensor 170, so the acquired image contains almost no high spatial frequency components. As a result, when the aforementioned spherical aberration occurs, the z position of the objective lens 104 where the contrast is maximized in the contrast curve of the image obtained by the sensor 163 deviates from the contrast curve of the image obtained by the two-dimensional image sensor 170. This deviation causes a positional shift in the detection result of the focus position in AF using the optical path length difference method.

[0056] Therefore, in this embodiment, the focusing control unit 140 controls the focus evaluation value calculation unit 164 to calculate a focus evaluation value for each of the images of multiple samples S acquired by the sensor 163 of the detection optical system, the images of which have different spatial frequency components. This focus evaluation value is calculated based on the contrast of each of the images of multiple samples S with different spatial frequency components. The focusing control unit 140 obtains the calculated focus evaluation value from the focus evaluation value calculation unit 164. Then, using the obtained focus evaluation value, the focusing control unit 140 estimates a focus evaluation value for the image of the sample S composed of predetermined spatial frequency components, more specifically, a focus evaluation value for the image of the sample S captured by the two-dimensional image sensor 170 of the observation optical system. Based on the focus evaluation value obtained by this estimation, the focusing control unit 140 controls the focusing unit 150 to move the sample S to the focus of the objective lens 104 and achieve focus.

[0057] Next, we will explain the method for estimating the focus evaluation value, referring to Figure 7.

[0058] The focus evaluation value calculation unit 164 first acquires the luminance value of light from the sample S detected by each pixel in the sensors 163 (first region 163a and second region 163b) located in the front and rear focus areas, respectively. At this time, the focus evaluation value calculation unit 164 sums the acquired luminance values ​​in the order of the pixel positions in order to bin a predetermined number of adjacent pixels into one pixel, and uses the resulting sum as the luminance value for the binned pixel. This binning process is the same as explained using Figure 4, but when estimating the focus evaluation value, the binning process is performed multiple times by changing the number of adjacent pixels binned into one pixel. In the example in Figure 7, the binning process is shown for cases where 2 adjacent pixels and 4 adjacent pixels are binned into one pixel. Therefore, the example in Figure 7 shows the case where three types of luminance values ​​are obtained: the luminance value when no binning is performed, the average of 1 pixel, the average of 2 pixels, and the average of 4 pixels. Therefore, when these brightness values ​​are arranged side by side, images of sample S with different degrees of blur are obtained, similar to images captured by image sensors with different pixel sizes. In other words, these images can be said to be images of sample S to which low-pass filters with different cutoff frequencies have been applied, that is, images of sample S with different spatial frequency components.

[0059] Next, the focus evaluation value calculation unit 164 calculates the contrast from three types of brightness values: 1 pixel, 2-pixel average, and 4-pixel average, and uses the calculated contrast to calculate the focus evaluation value.

[0060] The graph [A] in Figure 7 shows examples of contrast curves at the z position of the objective lens 104 and at the front and back focus positions. The solid curve represents the contrast curve obtained from the luminance value of each individual pixel without binning. The dashed-dotted curve represents the contrast curve obtained from the average luminance value of two adjacent pixels obtained by binning two adjacent pixels. The dotted curve represents the contrast curve obtained from the average luminance value of four adjacent pixels obtained by binning four adjacent pixels. Therefore, these curves can be said to be contrast curves for images of sample S with different spatial frequency components.

[0061] Furthermore, graph [B] in Figure 5 shows the relationship between the z position of the objective lens 104 and the focal evaluation value. In graph [B], the three curves—solid, dashed, and dotted—are graphs of the focal evaluation values ​​obtained from the solid, dashed, and dotted contrast curves in [A], respectively. These graphs can be said to be graphs of the focal evaluation values ​​for images of samples S with different spatial frequency components.

[0062] Note that graph [B] also shows a dashed curve. This curve is a graph of the focus evaluation value for the image of sample S obtained by the two-dimensional image sensor 170, estimated assuming that sensor 163 has the same pixel size as the two-dimensional image sensor 170. In this graph, the z position of the objective lens 104 where the focus evaluation value is 0 (zero) is the z position where the contrast of the image of sample S obtained by the two-dimensional image sensor 170 is maximum, as shown in the contrast curve [C], i.e., the precise focus position.

[0063] Furthermore, in graph [B], the black dots representing the focus evaluation value at the z position when the focus evaluation value shown by the solid curve is 0 (zero) are shown on each of the three curves: the solid line, the dashed line, and the dotted line. In this method, the focus evaluation value corresponding to the size of one pixel of the two-dimensional image sensor 170 is estimated based on the relationship between the sizes of one, two, and four pixels of the sensor 163, represented by these black dots, and the corresponding focus evaluation values.

[0064] Furthermore, when estimating the focal evaluation value, the z position of the objective lens 104 may be any position other than the position where the focal evaluation value is 0 (zero).

[0065] In this way, when estimating the focus evaluation value, the focusing control unit 140 first obtains the focus evaluation value for each of the images of multiple samples S, each having different spatial frequency components that constitute the image, from the focus evaluation value calculation unit 164. Then, using the obtained focus evaluation values, it estimates the focus evaluation value for the image of the sample S composed of predetermined spatial frequency components. More specifically, the focusing control unit 140 estimates the focus evaluation value for the image of the sample S obtained by the two-dimensional image sensor 170 when the z position of the objective lens 104 is at a predetermined position, from the focus evaluation values ​​shown in each of the three curves mentioned above. This estimation of the focus evaluation value is performed based on the relationship between the size of the pixels of the sensor 163 and the focus evaluation value. Alternatively, this estimation of the focus evaluation value may be performed based on the relationship between the size of the sample structure of interest and the focus evaluation value.

[0066] Subsequently, the focusing control unit 140 determines the direction and amount of movement of the objective lens 104 based on the estimated focus evaluation value, and controls the focusing unit 150 to move the focus of the objective lens 104 and focus on the sample S.

[0067] Here, an example of the procedure for AF control performed by the microscope system 1 will be explained according to the flowchart in Figure 8.

[0068] When this method is initiated, first, in step S101, the sensor 163 provided in the focusing unit 160 detects an image of the sample S at the front pin and rear pin positions, respectively.

[0069] Next, the focus control unit 140 controls the focus evaluation value calculation unit 164 to perform each process from step S102 to step S104.

[0070] First, in step S102, the focus evaluation value calculation unit 164 calculates the average or sum of the brightness values ​​of adjacent pixels of the sensor 163 multiple times while changing the number of adjacent pixels, thereby obtaining brightness values ​​for each of several different pixel sizes.

[0071] Next, in step S103, the focus evaluation value calculation unit 164 uses the brightness values ​​for each pixel size obtained in the process of step S102 to calculate the contrast of the image of the sample S at the front focus and back focus positions for each pixel size.

[0072] Next, in step S104, the focus evaluation value calculation unit 164 uses the contrast calculated in step S103 to calculate a focus evaluation value corresponding to each pixel size and outputs it to the focus control unit 140.

[0073] Next, in step S105, the focus control unit 140 uses the focus evaluation value calculated by the focus evaluation value calculation unit 164 to perform a process to estimate the focus evaluation value corresponding to the pixel size of the two-dimensional image sensor 170.

[0074] Next, in step S106, the focusing control unit 140 performs a process to determine the amount of drive (direction and movement) of the objective lens 104 based on the focus evaluation value estimated in the process of step S105.

[0075] Next, in step S107, the focusing control unit 140 controls the focusing unit 150 to move the objective lens 104 according to the drive amount determined in step S106.

[0076] Next, in step S108, the microscope control unit 110 performs a process to determine whether an instruction to terminate AF control has been input. If it is determined that an instruction to terminate AF control has been input (the determination result is YES), this AF control method is terminated. On the other hand, if it is determined that an instruction to terminate AF control has not been input (the determination result is NO), the process returns to step S101, and the above process is repeated until the focus evaluation value estimated in step S105 falls within the target range. At this time, if the estimated focus evaluation value is within the target range, the objective lens 104 is not driven, but the processes from step S101 to step S107 are repeated until an instruction to terminate AF control is input.

[0077] In microscope system 1, AF control is performed using the procedure described above.

[0078] Next, we will explain the method for estimating the focus evaluation value corresponding to the pixel size of the two-dimensional image sensor 170, which is performed by the focus control unit 140 as the processing of step S105 in the AF control method described above.

[0079] In this method, first, a function representing the relationship between multiple types of pixel sizes of the sensor 163 and the focus evaluation value corresponding to each pixel size, calculated by the focus evaluation value calculation unit 164, is obtained. Then, the focus evaluation value corresponding to the pixel size of the 2D image sensor 170 is obtained from this function and used as the estimation result.

[0080] The function described above can be said to represent the relationship between the spatial frequency components that constitute the image of sample S and the focal evaluation value for that image. Furthermore, the spatial frequency components that constitute the image of sample S captured by the two-dimensional image sensor 170 are components determined based on the size of the pixels of the two-dimensional image sensor 170. Therefore, the focal evaluation value obtained from this approximation curve, which corresponds to the pixel size of the two-dimensional image sensor 170, can be said to be the focal evaluation value for the image of sample S composed of predetermined spatial frequency components.

[0081] Furthermore, in this method, higher accuracy can be expected if a nonlinear function is used as the function representing the relationship between the pixel size of sensor 163 and the focus evaluation value. For example, an approximate curve obtained by function fitting may be used as this nonlinear function.

[0082] Figure 9 shows the first example of an approximate curve obtained by function fitting.

[0083] The approximation curve illustrated in Figure 9 is an example in which a second-order polynomial approximation curve was selected as the target of fitting. In this example, four different pixel sizes are used for the sensor 163 to obtain the approximation curve, and these pixel sizes (effective pixel sizes) are normalized to values ​​where the pixel size of the two-dimensional image sensor 170 is set to the reference value "1".

[0084] By using the approximation curve illustrated in Figure 9, the focus evaluation value corresponding to the pixel size of the 2D image sensor 170 is estimated to be "-5". The focusing control unit 140 then performs the process of determining the amount of drive (direction and movement) of the objective lens 104 according to this estimation result, as the process of step S106 in Figure 8 described above.

[0085] As described above, the microscope system 1 uses the focus evaluation values ​​for each of several images of the sample S, each having different spatial frequency components, to estimate the focus evaluation value for an image of the sample S composed of predetermined spatial frequency components. Based on the result of this estimation, the focusing unit is controlled. Therefore, this microscope system 1 can provide high-speed and high-precision autofocus (AF).

[0086] Furthermore, the configuration and control method of the microscope system 1 described above may be modified as described below.

[0087] For example, in the microscope system 1 described above, the amount of drive of the objective lens 104 is determined from the estimation result of the focus evaluation value for the image of the sample S captured by the two-dimensional image sensor 170, and control is performed to move the objective lens 104 according to the determined amount of drive. Alternatively, the z position of the objective lens 104 at which this focus evaluation value is a value indicating focus (i.e., 0 (zero)) may be determined from the estimation result of the focus evaluation value, and the microscope system 1 may perform control to move the objective lens 104 to this determined z position. Furthermore, the z position of the objective lens 104 at which the focus evaluation value for the image of the sample S captured by the two-dimensional image sensor 170 is a value indicating focus, i.e., the focus position, may be directly estimated, and the microscope system 1 may perform control to move the objective lens 104 to this focus position. Next, the method for estimating the focus position will be explained with reference to Figure 10.

[0088] The graphs [A], [B], and [C] shown in Figure 10 are similar to those shown in Figure 7. However, in graph [B], the black dots representing the z position of the objective lens 104 when the focus evaluation value is 0 (zero) are shown on three curves: a solid line, a dashed line, and a dotted line. In this method, the z position corresponding to the size of one pixel of the two-dimensional image sensor 170 is estimated based on the relationship between the sizes of one, two, and four pixels of the sensor 163 represented by these black dots and the corresponding z positions.

[0089] In this way, when estimating the focus position, the focus control unit 140 first obtains focus evaluation values ​​from the focus evaluation value calculation unit 164 for each of the images of multiple samples S whose spatial frequency components constituting the image are different from each other. Then, using the z position of the objective lens 104 when the obtained focus evaluation value is a value indicating focus, it estimates the z position of the objective lens 104 when the image of the sample S, composed of predetermined spatial frequency components, is in focus. More specifically, the focus control unit 140 estimates the z position of the objective lens 104 when the image of the sample S obtained by the two-dimensional image sensor 170 is in focus from the z position of the objective lens 104 shown in each of the three curves mentioned above. This z position estimation is performed based on the relationship between the size of the pixels of the sensor 163 and the z position of the objective lens 104 when the focus evaluation value is a value indicating focus.

[0090] Subsequently, the focusing control unit 140 determines the direction and amount of movement of the objective lens 104 based on the estimated z position, and controls the focusing unit 150 to move the focal point of the objective lens 104 and focus on the sample S.

[0091] Furthermore, the z-position of the objective lens 104 when the image of the sample S obtained by the two-dimensional image sensor 170 is in focus can be estimated using an approximation curve, for example, similar to the method for estimating the focus evaluation value described above. In this case, first, an approximation curve representing the relationship between multiple types of pixel sizes of the sensor 163 and the z-position when the focus evaluation value corresponding to each pixel size calculated by the focus evaluation value calculation unit 164 is a value indicating focus (i.e., 0 (zero)) is obtained. Then, the z-position corresponding to the pixel size of the two-dimensional image sensor 170 is obtained from this approximation curve and used as the result of estimating the focus position.

[0092] In this method as well, the approximate curve representing the relationship between the pixel size of sensor 163 and the z position when the focus evaluation value indicates focus may be obtained by function fitting.

[0093] Figure 11 shows a second example of an approximate curve obtained by function fitting.

[0094] The approximation curve illustrated in Figure 11 is an example where a logarithmic approximation curve was selected as the target for fitting. In this example, four different pixel sizes are used for the sensor 163 to obtain the approximation curve, and these pixel sizes are normalized to values ​​where the pixel size of the 2D image sensor 170 is set to the reference value "1".

[0095] By using the approximation curve illustrated in Figure 11, the z position of the objective lens 104 corresponding to the pixel size of the two-dimensional image sensor 170 is estimated to be "19872.4". The focusing control unit 140 controls the focusing unit 150 according to this estimation result to move the objective lens 104 to the estimated z position.

[0096] As described above, autofocus (AF) may be achieved by estimating the z position of the objective lens 104 at which the focus evaluation value for the image of the sample S captured by the two-dimensional image sensor 170 indicates focus, and then controlling the movement of the objective lens 104 to this estimated position using the microscope system 1.

[0097] Furthermore, the estimation of the focus evaluation value performed in the microscope system 1 is carried out using the relationship between the pixel sizes of the sensor 163 and the two-dimensional image sensor. If the difference in size between these two is excessively large, a significant decrease in estimation accuracy will occur. Therefore, the relationship between these two sizes may be made to satisfy the following equation [Equation 2].

[0098]

number

[0099] In equation [Equation 2], Po is the pixel size of the 2D image sensor 170, MGo is the imaging magnification of the observation optical system, Pd is the pixel size of the sensor 163, and MGd is the imaging magnification of the detection optical system.

[0100] Furthermore, since the image of sample S detected by sensor 163 is obtained by imaging the light from sample S that reaches through the detection optical system of microscope system 1, the spatial frequency components constituting this image should be components with spatial frequencies below the optical cutoff frequency of the detection optical system. This remains true even if the pixel size of sensor 163 is small enough to detect an image composed of spatial frequency components higher than this optical cutoff frequency.

[0101] Therefore, when calculating the focus evaluation value corresponding to each pixel size of the sensor 163, the pixel size may be determined based on the optical cutoff frequency of the detection optical system.

[0102] For example, if the NA of the objective lens 104, which is the detection optical system, is set to 0.4, the imaging magnification MGo of the detection optical system is set to 40 times, and the observation wavelength λ in visual observation is 550 nm (nanometers), then the optical cutoff frequency fc of the detection optical system will be the value of equation [Equation 3] below.

[0103]

number

[0104] On the other hand, if the pixel size Sp of one pixel of sensor 163 is 3.5 μm (micrometers), the Nyquist frequency fn in sampling by sensor 163 is the value of equation [Equation 4] below.

[0105]

number

[0106] At this time,

number

[0107] As described above, the focusing control unit 140 obtains focus evaluation values ​​from the focus evaluation value calculation unit 164 for each of the images of multiple samples S, each of which has different spatial frequency components constituting the image and whose spatial frequency components are lower than the cutoff frequency of the detection optical system. Then, using the obtained focus evaluation values, it estimates the focus evaluation value for the image of a sample S composed of predetermined spatial frequency components. By doing so, the use of information that could not be obtained in the estimation is eliminated, and an improvement in estimation accuracy can be expected.

[0108] Furthermore, in the method for estimating the focus evaluation value described above, the target for calculation of the focus evaluation value by the focus evaluation value calculation unit 164 may be selected based on spatial frequency component information of the image of the sample S, and the focus evaluation value calculated according to this selection may be acquired by the focus control unit 140. Next, this selection method will be explained with reference to Figure 12.

[0109] In Figure 12, the graphs arranged vertically on the left side show an example of the brightness values ​​of the image of the sample S detected by the front pin of the first region 163a of the line sensor sensor 163. In these graphs, the horizontal axis represents the y-direction position of each pixel, and the vertical axis represents the brightness value detected by the pixel at the corresponding y-direction position.

[0110] In Figure 12, the upper left graph shows two types of graphs: one for the brightness value of each individual pixel, and another for the average brightness value of eight adjacent pixels due to binning. These are represented by solid and dashed lines, respectively. The lower left graph in Figure 12 shows three types of graphs: one for the brightness value of each individual pixel, another for the average brightness value of 32 adjacent pixels due to binning, and a third for the average brightness value of 64 adjacent pixels. These are represented by solid, dashed, and dotted lines, respectively. In other words, the graphs arranged vertically on the left side of Figure 12 represent the spatial frequency component information of the image of sample S.

[0111] On the other hand, the graph on the right in Figure 12 is a curve showing an example of the relationship between the number of adjacent pixels used for averaging luminance values ​​by binning and the PV value of the average luminance value. "PV" is an abbreviation for Peak to Valley.

[0112] The higher the PV value, the better the image structure can be represented; a lower value indicates that the image structure is insufficient. Therefore, a threshold value is set for this PV value, and the number of adjacent pixels with a PV value greater than this threshold is used as the number of adjacent pixels for calculating the average brightness value.

[0113] For example, in the graph on the right side of Figure 12, if the threshold for the PV value is set to, for example, "20.0", then the number of adjacent pixels to be used for calculating the average brightness value is set to 8 pixels or less, which are evaluated as being able to represent the image structure in detail. In other words, in this case, the focus evaluation value calculation unit 164 selects four types: 1 pixel, 2-pixel average, 4-pixel average, and 8-pixel average, calculates the contrast for each of these four types of brightness values, and uses the calculated contrast to calculate the focus evaluation value. The focus control unit 140 obtains the four types of focus evaluation values ​​calculated in this way from the focus evaluation value calculation unit 164, and uses the obtained focus evaluation value to estimate the focus evaluation value for the image of the sample S captured by the 2D image sensor 170.

[0114] Note that the graphs arranged vertically on the left side of Figure 12 represent the brightness values ​​of the image of sample S detected by the first region 163a of sensor 163 at the front pin. However, the above selection may also be made using the brightness values ​​of the image of sample S detected by the second region 163b of sensor 163 at the back pin.

[0115] As described above, the focus evaluation value calculation unit 164 calculates a focus evaluation value for each of the multiple images of sample S, each composed of a plurality of spatial frequency components selected based on spatial frequency component information of the image of sample S at the front-pin or back-pin position. The focus control unit 140 then obtains the plurality of focus evaluation values ​​calculated in this way from the focus evaluation value calculation unit 164 and uses the obtained focus evaluation values ​​to estimate the focus evaluation value for the image of sample S captured by the two-dimensional image sensor 170. The focus evaluation value may be estimated in this manner.

[0116] Alternatively, spatial frequency component information for the image of sample S may be obtained using Fourier transform.

[0117] In Figure 13, the graph on the left, similar to the graph shown in Figure 12, shows an example of the brightness value of the image of the sample S detected by the first region 163a of the line sensor sensor 163 at the front pin. In this graph, the horizontal axis represents the y-direction position of each pixel, and the vertical axis represents the brightness value detected by the pixel at the corresponding y-direction position. The size of one pixel of this sensor 163 is assumed to be 14 μm.

[0118] On the other hand, the waveform on the right side of Figure 13 represents the spatial frequency component information of the image of sample S, obtained by performing a Fourier transform on the image information of sample S shown in the graph on the left side. Note that the amplitude of the leftmost part of this waveform, i.e., the part where the spatial frequency is close to 0, corresponds to the background of the image and is unrelated to the image of sample S.

[0119] Referring to this waveform, it can be seen that the image of sample S has spatial frequency components with a spatial frequency of approximately 0.065 [ / μm] or higher. The reciprocal of a spatial frequency of 0.065 [ / μm] is approximately 15.38 [μm], and this value is roughly the size of one pixel of sensor 163. Therefore, in this case, the focus evaluation value calculation unit 164 selects one of two types: one pixel and the average of two pixels.

[0120] As another example, let's consider the case where the image of sample S has spatial frequency components with a spatial frequency of approximately 0.02 [ / μm] or higher. The reciprocal of a spatial frequency of 0.065 [ / μm] is 50.00 [μm], and this value is approximately the size of four pixels of sensor 163. Therefore, in this example, the focus evaluation value calculation unit 164 selects one of three types: a single pixel, a two-pixel average, and a four-pixel average.

[0121] Thus, spatial frequency component information for the image of sample S may be obtained using the Fourier transform.

[0122] Furthermore, in the aforementioned microscope system 1, a function representing the relationship between multiple types of pixel sizes of sensor 163 and the focal evaluation value corresponding to each pixel size is obtained, and this obtained function is used to estimate the focal evaluation value corresponding to the pixel size of the 2D image sensor 170. It has been explained that this function may also be replaced with an approximation curve obtained by function fitting. Here, the focal evaluation value corresponding to each pixel size may be weighted, and a function representing the relationship between multiple types of pixel sizes of sensor 163 and the weighted evaluation value corresponding to each pixel size may be obtained, and this function may be used to estimate the focal evaluation value. This method will be explained with reference to Figures 14 and 15.

[0123] Figure 14 shows the first example of an approximate curve obtained by function fitting to weighted values.

[0124] In function fitting, the fitting function E(m) that minimizes the value Err obtained by equation [Equation 6] below is found.

[0125]

number

[0126] In equation [Equation 6], m and Em are the pixel size of the sensor 163 and the focus evaluation value calculated by the focus evaluation value calculation unit 164 for pixels of that pixel size, respectively, and Wm is the weight value assigned to the focus evaluation value.

[0127] As mentioned earlier, if no weighting is applied, the value of the weight Wm is always "1.0", regardless of the pixel size m. In contrast, when obtaining an approximate curve by function fitting to a weighted value, the value of the weight Wm is set to a value between 0 and 1.0 depending on the pixel size m.

[0128] Specifically, for example, if the value of m is 6 or less, the value of Wm is set to "1.0", and if the value of m is greater than 6, the value of Wm is set to "0.1". In this way, the focus evaluation value is estimated using an approximate curve obtained by function fitting, in which the weight of the focus evaluation value for the pixel size of sensor 163, which differs significantly from the pixel size of the 2D image sensor 170, is reduced. By doing so, it is expected that the accuracy of estimating the focus evaluation value corresponding to the pixel size of the 2D image sensor 170 will be improved.

[0129] Furthermore, the weight values ​​used in this weighting process may be set based on the spatial frequency component information of the image of sample S at the front pin (first position) or back pin (second position). In other words, the weights may be set using a curve that shows an example of the relationship between the number of adjacent pixels and the PV value of the average brightness value, as shown in the graph on the right side of Figure 12, which is used for averaging brightness values ​​by binning.

[0130] For example, if the PV value threshold is set to "20.0" in the graph on the right side of Figure 12, the weight for the focus evaluation value for adjacent pixel sizes of 8 pixels or less will be set to a value heavier than the weight for the focus evaluation value for adjacent pixel sizes of 8 pixels or less.

[0131] In this way, by setting a heavier weight on the focus evaluation value for pixel sizes that are evaluated as being able to represent the image structure in detail, it is expected that the accuracy of estimating the focus evaluation value corresponding to the pixel size of the 2D image sensor 170 can be improved.

[0132] Alternatively, spatial frequency component information for the image of sample S used to set the weight values ​​may be obtained using a Fourier transform.

[0133] For example, as mentioned above, the waveform on the right side of Figure 13 represents the spatial frequency component information of the image of sample S obtained by performing a Fourier transform on the image information of sample S obtained by sensor 163, where the size of one pixel is 14 μm (graph on the left). Referring to this waveform, it can be seen that the image of sample S has spatial frequency components with a spatial frequency of approximately 0.065 [ / μm] or more. The reciprocal of a spatial frequency of 0.065 [ / μm] is approximately 15.38 [μm], and this value is approximately the size of one pixel of sensor 163. Therefore, in this case, the focus evaluation value calculation unit 164 sets the weight for the focus evaluation value for the pixel size of one pixel and the weight for the focus evaluation value for the pixel size of two pixels to be heavier than the weight for the focus evaluation value for other pixel sizes. Even by setting the weights in this way, an improvement in the accuracy of estimating the focus evaluation value corresponding to the pixel size of the two-dimensional image sensor 170 can be expected.

[0134] Furthermore, as mentioned above, weighting may also be used when obtaining an approximate curve representing the relationship between the pixel size of the sensor 163 and the z position when the focus evaluation value is a value indicating focus, by function fitting. That is, weighting is applied to the z position when the focus evaluation value corresponding to the pixel size of the sensor 163, calculated by the focus evaluation value calculation unit 164, is a value indicating focus (i.e., 0 (zero)). Then, an approximate curve representing the relationship between the pixel size of the sensor 163 and the weighted z position is obtained by function fitting. Figure 15 shows a second example of an approximate curve obtained by function fitting to the weighted value in this way. Using the approximate curve obtained in this way, the z position corresponding to the pixel size of the 2D image sensor 170 may be obtained from this approximate curve as the estimated focus position.

[0135] The embodiments described above are merely examples to facilitate understanding of the invention, and the present invention is not limited to these embodiments. Modified forms of the embodiments described above and alternative forms that replace the embodiments described above may be included. In other words, the components of the embodiments described above can be modified without departing from the spirit and scope thereof. Furthermore, new embodiments can be implemented by appropriately combining multiple components disclosed in the embodiments. In addition, some components may be deleted from the components shown in the embodiments, or some components may be added to the components shown in the embodiments. Moreover, the processing procedures shown in the embodiments may be performed in a different order, as long as it does not contradict the original. That is to say, the microscope system of the present invention can be modified and changed in various ways without departing from the scope of the claims.

[0136] In this specification, the expression "based on A" does not mean "based solely on A," but rather "based on at least A," and furthermore, "based at least partially on A." In other words, "based on A" may also be based on B in addition to A, or on a part of A. [Explanation of Symbols]

[0137] 1. Microscope System 10. Actual field of view 100 Microscopes 101 Light source 102 Condenser Lens 103 Stages 104 Objective lens 105, 162 splitter 106 Imaging lens 110 Microscope Control Unit 111, 121, 131, 141 Control circuits 120 Light source control unit 130 Stage Control Unit 140 Focusing Control Unit 150 Aiming section 160 Focusing Unit 161 Focusing lens 163 Sensors 163a 1st area 163b 2nd area 164 Focus Evaluation Value Calculation Unit 170 2D image sensor 180 Control Unit 200 Image Processing Devices S sample CG cover glass SG microscope slides

Claims

1. The stage and, The objective lens, A focusing unit that changes the distance between the stage and the objective lens, A branching section that splits the light from the sample supported on the stage, which has passed through the objective lens, into the optical axis of the observation optical system and the optical axis of the detection optical system, A sensor that acquires brightness values ​​for the image of the sample at a first position and a second position on the optical axis of the detection optical system, where the first position is in front of the position optically conjugate to the front focal point of the objective lens and the second position is behind the position, respectively. A focus evaluation value calculation unit calculates a focus evaluation value for the image of the sample based on the contrast obtained from the luminance values ​​at the first position and the second position, respectively, acquired by the sensor. An image sensor for capturing an image of the sample formed on the optical axis of the observation optical system, A control unit, The focus evaluation value for each of the images of the multiple samples, each having different spatial frequency components that constitute the image of the sample, is obtained from the focus evaluation value calculation unit. Using the acquired focus evaluation value, the focus evaluation value for the image of the sample composed of predetermined spatial frequency components is estimated. The focusing unit is controlled based on the results of the above estimation. The control unit and A microscope system characterized by being equipped with the following features.

2. The microscope system according to claim 1, characterized in that a transmitted illumination optical system for illuminating the sample is provided on the opposite side of the objective lens, across the stage.

3. The microscope system according to claim 1, characterized in that the objective lens has a numerical aperture of 0.3 or more.

4. The microscope system according to claim 1, characterized in that the control unit performs the estimation using an approximation curve representing the relationship between the spatial frequency components constituting the image of the sample and the focal evaluation value for the image of the sample.

5. The sensor includes a photodetector that detects the light at the first position and the second position, When the pixel size of the image sensor is Po, the imaging magnification of the observation optical system is MGo, the pixel size of the photodetector is Pd, and the imaging magnification of the detection optical system is MGd, then the following conditions apply. [Number 7] The microscope system according to claim 1, characterized in that it satisfies the following conditions.

6. The microscope system according to claim 1, characterized in that the control unit obtains the focus evaluation value for each of the images of a plurality of samples, each composed of spatial frequency components lower than the cutoff frequency of the detection optical system, from the focus evaluation value calculation unit.

7. The microscope system according to claim 1, characterized in that the control unit obtains the focus evaluation value for each of the multiple images of the sample, each composed of each of the multiple spatial frequency components selected based on spatial frequency component information of the image of the sample at the first position or the second position, from the focus evaluation value calculation unit.

8. The microscope system according to claim 1, characterized in that the control unit performs the estimation using an approximation curve representing the relationship between the spatial frequency components constituting the image of the sample and the evaluation value obtained by weighting the focal evaluation value for the image of the sample.

9. The microscope system according to claim 8, characterized in that the weights in the weighting are set based on spatial frequency component information of the image of the sample at the first position or the second position.

10. The microscope system according to any one of claims 1 to 9, characterized in that the image of the sample composed of the predetermined spatial frequency components is an image of the sample captured by the image sensor.

Images