Microscope, observation methods

Abstract

To provide a microscope with an autofocus mechanism that achieves both focus accuracy and an increase in the speed of focus follow-up.SOLUTION: A microscope according to the present invention moves the position of a collimator lens so that light passing through an objective lens is focused on a surface of a cover glass, and detects reflected light from a sample as a focus error signal representing the amount of deviation of a focal point of the light passing through the objective lens from a focus target surface.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a microscope.

Background Art

[0002] Pathology is a field of medicine that diagnoses diseases by examining tissues, surgically removed organs, body fluids, etc. under a microscope. In recent years, with the spread of digital images, especially the development and spread of WSI (Whole Slide Imaging) technology, traditional pathology has evolved into digital pathology. WSI is a system that first captures an image of the entire glass slide specimen using a high-magnification microscope in advance, imports it into a computer as a digital image, and displays it on a monitor device for observation. The imaging device for digital images is called a WSI scanner.

[0003] In many applications of digital pathology, the scanning process of specimens has become the main bottleneck in the workflow, and an improvement in the throughput of the scanning process (reduction of scanning time) is required.

[0004] By the way, in the microscope of a WSI scanner, autofocus control is performed to automatically adjust the focus of the objective lens to the optimal position in order to obtain a clear image of the object. The autofocus method for WSI scanners is roughly classified into two types: a reflection type and an image-based type from the perspective of the focus detection method. The reflection type uses a dedicated light source to receive the reflected light from the sample and detects the height of the reference surface on the sample. The image-based type detects the focus height based on the image quality evaluation value (contrast, etc.) of the captured image.

[0005] Astigmatism, a type of reflective focus detection method, detects focus shift (defocus) by focusing a laser beam onto the focus detection target surface and receiving the reflected light. The detection system in this method consists of a focusing lens, a cylindrical lens, and a detector, and detects defocus by utilizing the change in the shape of the light spot on the detector according to the amount of defocus. Because this astigmatism method uses a light source and detection system dedicated to focus detection, it does not require image processing of captured images, as is the case with image-based focus detection methods. Furthermore, because it can be implemented with simple signal processing, it excels in high-speed focus tracking and is a promising method for reducing the scan time of WSI scanners.

[0006] Patent Document 1 below describes an example of an autofocus device using the astigmatism method. The document aims to "provide an autofocus device and microscope device that can shorten the time required to focus the objective lens on an object," and describes the technology as follows: "The autofocus device 20 comprises a drive unit 25 that moves the objective lens 11 in the optical axis direction, a detection unit 28 that detects whether or not an object is placed at a detection position DP shifted by a predetermined reference distance from the focal plane of the objective lens 11, and a control unit 29 that, when the detection unit 28 detects that an object is placed at the detection position DP, drives the drive unit 25 to move the objective lens 11 by the reference distance and controls the focus of the objective lens 11 on the object." (See abstract). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-137838 [Overview of the project] [Problems that the invention aims to solve]

[0008] One form of glass slide specimen used as the imaging target for a WSI scanner is a tissue section placed on a glass slide and then mounted with a coverslip using a mounting medium. For the astigmatism-based focus detection method to function ideally, that is, with high focus detection sensitivity, it is necessary to focus the laser beam for focus detection (hereinafter referred to as the focus detection beam) onto the surface to be focused. When applying the astigmatism method to autofocus on a glass slide specimen, the laser beam onto the specimen is performed using the objective lens of a microscope, and the surface to be focused is the main reflective surface of the irradiated light, i.e., the surface of the coverslip.

[0009] However, when imaging tissue sections, the position (height) of the objective lens must be adjusted to focus on the tissue section, which imposes a constraint. Therefore, it is not always possible to focus the focus detection beam onto the cover glass surface, which is the target of the focus. In most cases, the focus detection beam is a parallel beam, and the point of focus by the objective lens is the position of the tissue section. Consequently, the beam spreads out on the cover glass surface, and the focus detection sensitivity, i.e., the change in the focus error signal relative to the amount of defocus, decreases significantly compared to the ideal case. This decrease in focus detection sensitivity leads to a decrease in focus accuracy.

[0010] The objective of the present invention is to solve the above problems and provide a microscope with an autofocus mechanism that achieves both high-speed focus tracking and high focus accuracy. [Means for solving the problem]

[0011] The microscope according to the present invention moves the position of the collimator lens so that the light that has passed through the objective lens is focused on the surface of the cover glass, and detects the reflected light from the sample as a focus error signal that represents the amount of deviation of the focal point of the light that has passed through the objective lens from the plane of focus. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a microscope with an autofocus mechanism that achieves both high-speed focus tracking and high focus accuracy. Other problems, configurations, and advantages of the present invention will become clear from the following description of embodiments. [Brief explanation of the drawing]

[0013] [Figure 1] An example of the configuration of the imaging optical system included in the microscope according to Embodiment 1 is shown. [Figure 2] This diagram illustrates the principle of detecting focus error, i.e., the amount of deviation of the focal point of the illumination beam from the target plane, using the astigmatism method. [Figure 3] This diagram schematically shows one form of the focusing state of the focus detection beam 139. [Figure 4] This figure shows the shape of the light spot on the photodetector 153 when the position of the objective lens 121 is changed, with the state in Figure 3 being set as defocus = 0, and this is used as a reference. [Figure 5] This graph plots the relationship between defocus and the focus error signal (FES) when the state in Figure 3 is set to defocus = 0. [Figure 6] This diagram schematically shows another form of the focusing state of the focus detection beam 139. [Figure 7] This figure shows the shape of the light spot on the photodetector 153 when the position of the objective lens 121 is changed, with the state in Figure 6 being set as defocus = 0, and this is used as a reference. [Figure 8] This graph plots the relationship between defocus and the focus error signal (FES) when the state in Figure 6 is set to defocus = 0. [Figure 9] This diagram schematically shows the focusing state of the focus detection beam 139. [Figure 10] This figure shows the shape of the light spot on the photodetector 153 when the position of the objective lens 121 is changed, with the state in Figure 9 being set as defocus = 0, and this is used as a reference. [Figure 11]A graph plotting the relationship between defocus and focus error signal (FES) when the state of FIG. 9 is set to defocus = 0. [Figure 12] A flowchart explaining the procedure for performing autofocus control by adjusting the position of the collimator lens 132 according to an arbitrary glass slide specimen. [Figure 13] A conceptual diagram showing the principle by which a focus error occurs due to unevenness in the cover glass thickness. [Figure 14] A top view showing an example of the arrangement of focus learning points. [Figure 15] A flowchart explaining the procedure for performing autofocus control using a focus error signal corrected by creating a focus error signal correction value map and using the data thereof. [Figure 16] A flowchart explaining the procedure for performing autofocus control while dynamically controlling the position of the collimator lens 132 based on data by creating a collimator lens position map. [Figure 17] A diagram showing a configuration example of an imaging optical system included in the microscope according to Embodiment 6.

Mode for Carrying Out the Invention

[0014] <Embodiment 1: Basic Configuration (Adjustment of Beam Focus Point Position)> <Configuration of the Entire Optical System> FIG. 1 shows a configuration example of an imaging optical system included in the microscope according to Embodiment 1 of the present invention. This imaging optical system includes a microscope optical system 1 for imaging a specimen, a focus detection optical system 2 for detecting a focus error signal for performing autofocus, and an arithmetic unit 200. The coordinate axes are defined such that the optical axis direction of the microscope optical system 1 is the Z-axis, the horizontal direction in the plane of the glass slide specimen described later with respect to the paper surface is the X-axis, and the vertical direction with respect to the paper surface is the Y-axis. The arithmetic unit 200 controls each part of the microscope and executes the flowchart described later.

[0015] <Configuration of the Microscope> The configuration of the microscope optical system 1 is classified as a transmission bright-field microscope. The illumination optical system 3 for illuminating the object to be imaged comprises a light-emitting diode 101 as a light source, a collector lens 102, and a condenser lens 103. The collector lens 102 creates a real image of the light source on the condenser lens 103, and the condenser lens 103 creates a real image of the collector lens 102 on the sample surface. In other words, the illumination method of the illumination optical system 3 is Köhler illumination, which uniformly illuminates the sample surface. The illumination optical system 3 also includes a field diaphragm 104 and an aperture diaphragm 105. The field diaphragm 104 adjusts the illumination range on the sample surface, and the aperture diaphragm 105 adjusts the numerical aperture of the illumination light.

[0016] The glass slide specimens used for imaging consist of tissue sections placed on a glass slide and then mounted using a mounting medium and a coverslip 112.

[0017] The objective lens 121 and the imaging lens 123 constitute an infinity-corrected optical system, which forms an image of the tissue section in the glass slide specimen onto the sensor surface of the image sensor 124. The objective lens position adjustment mechanism 122 adjusts the position of the objective lens 121 in the optical axis direction.

[0018] <Configuration of the focus detection optical system> The focus detection optical system 2 detects the focus error based on the astigmatism method. The laser diode 131, which is the light source, emits linearly polarized infrared laser light with a wavelength of 785 nm. The emitted laser light is collimated by the collimator lens 132 to become a nearly parallel beam, and then the beam diameter is limited to a predetermined size by the aperture 134 to become the focus detection beam 135. The focus detection beam 135 passes through the λ / 2 plate 136 and is incident on the polarized beam splitter 137.

[0019] The polarizing beam splitter 137 has the function of transmitting almost 100% of p-polarized light (polarization in which the electric field component is parallel to the incident surface) and reflecting almost 100% of s-polarized light (polarization in which the electric field component is perpendicular to the incident surface) incident on the separation surface. By adjusting the rotation angle of the λ / 2 plate 136 around the optical axis with respect to the focus detection beam 135, the polarization direction of the light transmitted through the λ / 2 plate 136 can be arbitrarily changed, and the intensity ratio of transmitted light to reflected light at the polarizing beam splitter 137 can be arbitrarily adjusted. In this embodiment, the rotation angle of the λ / 2 plate 136 is adjusted so that the light transmitted through the λ / 2 plate 136 is p-polarized, and the focus detection beam 135 is configured to transmit almost 100% of the polarizing beam splitter 137.

[0020] The focus detection beam 135, which has been transmitted almost 100% through the polarizing beam splitter 137, is converted to circular polarization by passing through a λ / 4 plate 138, which is positioned with its velocity axis tilted at 45 degrees relative to the polarization direction of the incident beam, and is then guided to the microscope optical system 1. The focus detection beam 135 is coupled to the optical path of the microscope optical system 1 by a dichroic filter 141 placed in the optical path of the microscope optical system 1. Subsequently, the focus detection beam 135 is focused by the objective lens 121 and irradiated onto the glass slide specimen, which is the object to be imaged.

[0021] The beam reflected from the surface of the cover glass 112 of the glass slide specimen (hereinafter referred to as the reflected light beam 139 or focus detection beam 139) is returned to a nearly parallel beam by the objective lens 121 and then converted back to linear polarization by the λ / 4 plate 138. At this time, the rotation direction of the circularly polarized light is reversed by the reflection from the surface of the cover glass 112, so the direction of the linearly polarized light becomes s-polarized, which is rotated 90 degrees from the direction of the forward light. Therefore, the s-polarized reflected light beam 139 that has passed through the λ / 4 plate 138 is reflected almost 100% by the polarizing beam splitter 137 and heads towards the focusing lens 151.

[0022] The reflected light beam 139 is focused by the focusing lens 151 and then incident on the cylindrical lens 152. The cylindrical lens 152 is positioned with its cylindrical axis tilted 45 degrees with respect to the Y axis in the XY plane. The reflected light beam 139 is transmitted through the cylindrical lens 152, resulting in astigmatism, and then incident on the photodetector 153. The photodetector 153 is a four-segment photodiode, and its division lines are positioned along the X and Y axes. A focus error signal (FES), described later, is generated from the output signals of each element (A, B, C, and D) of the photodetector 153.

[0023] <Principle of focus detection using astigmatism> Figure 2 illustrates the principle of detecting focus error, i.e., the amount of deviation of the focal point of the illumination beam from the target plane, using the astigmatism method.

[0024] The reflected light beam 139 is focused by the focusing lens 151, while astigmatism is added by the cylindrical lens 152. The cylindrical lens 152 acts as a lens in only one direction, and in the direction perpendicular to it, it behaves like a parallel plate and does not have a lens effect. Therefore, astigmatism occurs because the focal length differs in each direction. In the focus detection optical system 2 of this embodiment, the focal length of the beam viewed from the cross section where the cylindrical lens 152 does not have a lens effect is approximately the same as the focal length (fdet) of the focusing lens 151. On the other hand, the beam viewed from the cross section where the lens effect is present does not focus there, and an elongated line image (focused line) is obtained.

[0025] The beam viewed from the lensing cross-section of the cylindrical lens 152 converges to the focal length of the combined lens of the focusing lens 151 and the cylindrical lens 152. However, the beam viewed from the cross-section without lensing does not focus there and becomes a focal line. The focal length of the combined lens, fcomb, is expressed by Equation 1, where fdet is the focal length of the focusing lens 151, fcyl is the focal length of the cylindrical lens 152, and Dlens is the distance between the focusing lens 151 and the cylindrical lens 152. The beam becomes circular midway between the two focal lines.

[0026]

number

[0027] When measuring defocus using the astigmatism method, the center of the photodetector 153 is aligned to a position where the reflected light beam from the focal plane of the objective lens 121 is circular, as shown in Figure 2(2). The photodetector 153 is composed of four segmented photodiodes, and the optical signal detected by each element is converted into an electrical signal. The signal processing circuit calculates the focus error signal FES from the four outputs A, B, C, and D of the photodetector 153 using Equation 2.

[0028]

number

[0029] When the height of the surface of the cover glass 112, which is the focusing plane, coincides with the focal plane (focal length) of the objective lens 121, FES = 0. As shown in Figure 2(1), if the focusing plane is lower than the focal plane, the beam becomes elongated horizontally to the left, and FES > 0. On the other hand, as shown in Figure 2(3), if the focusing plane is higher than the focal plane, the beam becomes elongated horizontally to the right, and FES < 0.

[0030] The sign of FES depends on the installation angle of the cylindrical lens 152 with the optical axis direction as the rotation center. In this embodiment, it is installed at a 45-degree angle to the vertical with the optical axis direction as the rotation center. Here, the optical axis direction is defined as the direction in which light propagates. In this way, the amount of deviation from the cover glass surface is detected based on the focus error signal.

[0031] <Focus detection sensitivity> Figure 3 schematically shows one configuration of the focus detection beam 139's focused state. The mounting medium 302 is for mounting the tissue section 301. In this configuration, the focus detection beam 139 is a parallel beam, and the position of the objective lens 121 is adjusted so that the focus detection beam 139 is focused onto the surface of the cover glass 112. In other words, this is one configuration in which the astigmatism method functions ideally.

[0032] Figure 4 shows the shape of the light spot on the photodetector 153 when the position of the objective lens 121 is changed, with the state in Figure 3 being set as defocus = 0. Figures 4(1), 4(2), and 4(3) show the cases where the defocus is -20, 0, and +20 μm, respectively. The size of the photodetector was set to 0.5 mm × 0.5 mm. The plot area is the same as the size of the photodetector, 0.5 mm × 0.5 mm. As shown, the shape of the spot on the photodetector 153 changes diagonally and becomes elongated in response to the change in defocus.

[0033] Figure 5 is a graph plotting the relationship between defocus and the focus error signal (FES) when the state in Figure 3 is set to defocus = 0. As shown, the focus error signal changes linearly with respect to defocus, between the positive and negative peaks, indicating that the astigmatism method is functioning ideally.

[0034] However, in the state shown in Figure 3, the objective lens 121 is focused on the surface of the cover glass 112, so the original purpose of the WSI scanner, which is to obtain a clear image of the tissue section 301, cannot be achieved.

[0035] Figure 6 schematically shows another configuration of the focus detection beam 139's focusing state. The focus detection beam 139 is a parallel beam, as in the case of Figure 3. On the other hand, the position of the objective lens 121 is adjusted to focus on the tissue section 301. That is, this represents the actual operating state of the WSI scanner. However, in this state, the focus detection beam 139 is focused on the tissue section 301 and spreads out on the surface of the cover glass 112, which is the target surface for focusing.

[0036] Figure 7 shows the shape of the light spot on the photodetector 153 when the position of the objective lens 121 is changed, with the state in Figure 6 being set as defocus = 0 as the reference point. Figures 7(1), (2), and (3) show the cases where the defocus is -20, 0, and +20 μm, respectively. The plot area is 2.0 mm × 2.0 mm, unlike in Figure 3. The size of the photodetector (0.5 mm × 0.5 mm) is shown in the figure with a white dashed line. As shown, in the state in Figure 6, the light spot on the photodetector 153 spreads out widely and extends beyond the photodetector 153. As a result, the focus error signal hardly changes with respect to the change in defocus, making it difficult to detect defocus. Even if a photodetector large enough to receive the entire light spot is used, the change in the shape of the light spot with respect to the change in defocus is significantly smaller compared to the ideal state in Figure 3.

[0037] Figure 8 is a graph plotting the relationship between defocus and the focus error signal (FES) when the state in Figure 6 is set to defocus = 0. As can be seen, the focus error signal hardly changes around defocus = 0, meaning that the focus detection sensitivity is extremely low.

[0038] Figure 9 schematically shows the focusing state of the focus detection beam 139 in this embodiment. The position of the objective lens 121 is adjusted to focus on the tissue section 301. That is, it represents the actual operating state of the WSI scanner. However, unlike in Figure 3, the focus detection beam 139 is weakly focused as it enters the objective lens 121 and focuses on the surface of the cover glass 112, which is the focus target surface. With this configuration, it is possible to ideally enable focus detection by astigmatism while positioning the objective lens 121 in a position suitable for imaging.

[0039] Figure 10 shows the shape of the light spot on the photodetector 153 when the position of the objective lens 121 is changed, with the state in Figure 9 being set as defocus = 0. Figures 10(1), (2), and (3) show the cases where the defocus is -20, 0, and +20 μm, respectively. The plot area is 0.5 mm × 0.5 mm, the same as the size of the photodetector. As shown, in the state in Figure 9, the change in the shape of the light spot with respect to defocus is large and is equivalent to the change in the shape of the light spot in the configuration in Figure 3 (Figure 4).

[0040] Figure 11 is a graph plotting the relationship between defocus and the focus error signal (FES) when the state in Figure 9 is set to defocus = 0. As shown, the slope in the linear region between the positive and negative peaks of the focus error signal, i.e., the focus detection sensitivity, is equivalent to the focus error signal in the ideal state in Figure 3 (Figure 5). This indicates that focus detection by astigmatism functions ideally with the configuration of this embodiment.

[0041] <Adjusting the beam focus point> The focused state of the focus detection beam 139 shown in Figure 9, that is, the state in which the focus detection beam 139 is focused on the surface of the cover glass 112 with the focus of the objective lens 121 aligned with the tissue section 301, is achieved by the following means.

[0042] The position of the collimator lens 132 shown in Figure 1 is based on the position where the laser light transmitted through the collimator lens 132 becomes a parallel beam. The collimator lens position adjustment mechanism 133 moves the position of the collimator lens 132 along the optical axis in the direction of beam propagation. As a result, the laser light transmitted through the collimator lens 132 becomes a beam that propagates while being weakly focused. The weakly focused focus detection beam 135 is focused by the objective lens 121, and the position of its focus point is in front of the focal plane (focal length) of the objective lens 121.

[0043] The collimator lens position adjustment mechanism 133 adjusts the position of the collimator lens 132 so that the focus detection beam 135 is focused precisely on the surface of the cover glass 112.

[0044] <Embodiment 2: Method for adjusting the position of the beam focusing point> <Adjust to bring the focus error signal as close to zero as possible> Embodiment 2 of the present invention relates to a procedure for adjusting the collimator lens 132 to a position suitable for focus detection based on astigmatism. The configuration of the microscope is the same as in Embodiment 1. First, the procedure for setting the focus detection optical system 2 will be described. In the focus detection optical system 2 of Figure 1, the photodetector 153 is positioned so that the focus error signal becomes zero when the focus detection beam 135 is focused just onto the surface of the cover glass 112. An example of the procedure for positioning the photodetector 153 in such a position is shown below.

[0045] First, the position of the objective lens 121 is adjusted so that its focal plane coincides with the surface of the cover glass 112. This state is achieved, for example, by adjusting the position of the objective lens 121 while observing the image captured by the microscope optical system 1, so that minute scratches on the surface of the cover glass 112 or minute dust particles attached to the surface are clearly visible. Next, the photodetector 153 is placed at a position where the focus error signal becomes zero when the focus detection beam 135 is a parallel beam. That is, the photodetector 153 is placed at a position where the reflected light beam 139 is circular. Through these steps, the focus detection optical system 2 is set to a state where the focus error signal becomes zero when the focus detection beam 135 is focused precisely on the surface of the cover glass 112.

[0046] Figure 12 is a flowchart illustrating the procedure for performing autofocus control by adjusting the position of the collimator lens 132 to match any glass slide specimen. The focus detection optical system 2 is assumed to be pre-set to a position where the focus error signal becomes zero when the focus detection beam 135 is precisely focused on the surface of the cover glass 112. This flowchart (and the following flowcharts as well) is performed by the calculation unit 200. The steps in Figure 12 are described below.

[0047] Step S12: Focus the objective lens 121 on the tissue section at an arbitrary representative point (XY coordinate) on the glass slide specimen, preferably at the location where the tissue section to be imaged is located. That is, move the objective lens 121 to the position where the imaged image of the tissue section is clearest.

[0048] Step S13: The collimator lens position adjustment mechanism 133 moves the collimator lens 132 to a position where the focus error signal is zero. In this step, the focus detection beam 135 is focused onto the surface of the cover glass 112 at a representative point.

[0049] Step S14: This step is an autofocus control step. A focus error signal is detected, and feedback control is repeatedly performed at coordinates other than the representative point in S12 to move the objective lens 121 in a direction that brings the focus error signal closer to zero.

[0050] The above procedure is performed each time a glass slide specimen is replaced. This ensures that even if the height of the cover glass surface changes due to variations in the thickness of the slide glass and cover glass in individual glass slide specimens, the focus detection beam 135 can always be focused onto the surface of the cover glass 112, thereby ensuring high focus detection sensitivity.

[0051] <Embodiment 3: Focus Error Signal Correction Value Map> The astigmatism method detects the focus error relative to the cover glass surface by receiving reflected light from the cover glass surface. In autofocus using this method, the position of the objective lens is controlled based on the position on the cover glass surface. However, commercially available cover glasses have variations in thickness, that is, variations in thickness depending on the location within a single cover glass. As a result, the distance from the cover glass surface to the tissue section varies depending on the area being imaged, and this variation becomes a focus error. Embodiment 3 of the present invention relates to a method for suppressing focus errors caused by variations in cover glass thickness in glass slide specimens. The microscope configuration is the same as in Embodiment 1.

[0052] <Focus error due to uneven cover glass thickness> Figure 13 is a conceptual diagram illustrating the principle by which focus errors occur due to variations in the thickness of the cover glass. Figure 13 corresponds to a side cross-section of the cover glass. Autofocus control controls the position of the objective lens 121 to follow changes in the surface height of the cover glass 112. However, if the thickness of the cover glass 112 differs depending on the imaging location (XY coordinates), the distance from the surface of the cover glass 112 to the tissue section 301 being imaged changes, and this change results in a focus error.

[0053] <Creating a map, placing focus learning points> Figure 14 is a top view showing an example of the arrangement of focus learning points. The imaging range is set to include the entire tissue section 301 to be imaged, and focus learning points 401 are arranged at predetermined intervals within the imaging range. It is desirable that the arrangement interval of the focus learning points 401 be less than half of the reciprocal of the maximum spatial frequency of the thickness variation of the cover glass (minimum spatial period).

[0054] <interpolation> The correction value used to correct the focus error signal can be set for each XY coordinate on the surface of the tissue section 301. The data describing this correction value will be called the focus error signal correction value map. One form of the focus error signal correction value map is a lookup table that stores the correspondence between XY coordinates and focus error signal correction values ​​as an array. The focus error signal correction value for XY coordinates between focus learning points not included in the map is calculated by interpolation using data from multiple nearby focus learning points. Alternatively, the focus error signal correction value can be expressed as a mathematical formula for calculating it as a function of the XY coordinates.

[0055] <Flowchart> Figure 15 is a flowchart illustrating the procedure for creating a focus error signal correction value map and performing autofocus control using the focus error signal corrected by that data. The following describes each step in Figure 15.

[0056] Step S22: Focus the objective lens 121 on the tissue section at an arbitrary representative point (XY coordinates) on the glass slide specimen, preferably at the location where the tissue section to be imaged is located. That is, move the objective lens 121 to the position where the imaged image of the tissue section is clearest.

[0057] Step S23: The collimator lens position adjustment mechanism 133 moves the collimator lens 132 to a position where the focus error signal is zero. In this step, the focus detection beam 135 is focused onto the surface of the cover glass 112 at a representative point.

[0058] Step S24: Move to the position (XY coordinates) of the next focus learning point.

[0059] Step S25: At the focus learning point, the objective lens 121 is focused on the tissue section. That is, the objective lens 121 is moved to the position where the image of the tissue section is clearest.

[0060] Step S26: Detect the focus error signal and record it as the focus error signal correction value.

[0061] Step S27: Determine whether steps S25 and S26 have been performed for all focus learning points. If YES, proceed to the next step S28. If NO, return to step S24.

[0062] Step S28: Create a focus error signal correction value map that shows the relationship between XY coordinates and the focus error signal correction value at those coordinates.

[0063] Step S29: This step is an autofocus control step. A focus error signal is detected, and the focus error signal is corrected by subtracting the focus error signal correction value at the current XY coordinates from the detected focus error signal. Feedback control is repeatedly performed to move the objective lens 121 in a direction that brings the corrected focus error signal closer to zero.

[0064] <Embodiment 4: Map-Specific Options> <Flowchart> Embodiment 4 of the present invention describes a collimator lens position map created from a different perspective than the focus error signal correction value map described in Embodiment 3. The microscope configuration is the same as in Embodiment 1.

[0065] Figure 16 is a flowchart illustrating the procedure for creating a collimator lens position map and performing autofocus control while dynamically controlling the position of the collimator lens 132 based on that data. The following describes each step in Figure 16.

[0066] Step S32: Focus the objective lens 121 on the tissue section at an arbitrary representative point (XY coordinate) on the glass slide specimen, preferably at the location where the tissue section to be imaged is located. That is, move the objective lens 121 to the position where the imaged image of the tissue section is clearest.

[0067] Step S33: The collimator lens position adjustment mechanism 133 moves the collimator lens 132 to a position where the focus error signal is zero. In this step, the focus detection beam 135 is focused onto the surface of the cover glass 112 at a representative point.

[0068] Step S34: Move to the position (XY coordinates) of the next focus learning point.

[0069] Step S35: At the focus learning point, the objective lens 121 is focused on the tissue section. That is, the objective lens 121 is moved to the position where the image of the tissue section is clearest.

[0070] Step S36: Move the collimator lens 132 to a position where the focus error signal becomes zero, and record this position as the collimator lens position target value.

[0071] Step S37: Determine whether steps S35 and S36 have been performed for all focus learning points. If YES, proceed to the next step S38. If NO, return to step S34.

[0072] Step S38: Create a collimator lens position target value map that shows the relationship between XY coordinates and the collimator lens position target value at those coordinates.

[0073] Step S39: This step is an autofocus control step. The control of the position of the objective lens 121 is the same as in the above embodiment. That is, a focus error signal is detected, and feedback control is repeatedly performed to move the objective lens 121 in a direction that approaches zero.

[0074] In this fourth embodiment, the position of the collimator lens 132 is feedforward controlled to a target position corresponding to the XY coordinates. This target position is derived from the collimator lens position target value map. By adjusting the position of the collimator lens 132, the light beam can be irradiated in a more ideal state.

[0075] <Embodiment 5: Dedicated Map Creation Device> Embodiment 5 of the present invention relates to another form of creating the focus error signal correction value map of Embodiment 3 and the collimator lens position target value map of Embodiment 4. These maps may be created by another device (map creation device) having a configuration equivalent to that of the microscope according to the above embodiments. For example, the observation process can be carried out smoothly by having the map creation device create the map of the slide that the microscope will next observe in advance. The map creation device does not necessarily have to have exactly the same configuration as the microscope according to the present invention, as long as it has a configuration that can create the maps described in Embodiments 3 to 4.

[0076] <Embodiment 6: Dual Astigmatism Method> Figure 17 shows an example of the configuration of the imaging optical system included in a microscope according to Embodiment 6 of the present invention. Embodiment 6 relates to an alternative configuration of the focus detection optical system. The other configurations are the same as in the above embodiments.

[0077] The s-polarized reflected light beam 139 that has passed through the λ / 4 plate 138 is reflected almost 100% by the polarizing beam splitter 137 and heads toward the focusing lens 151. The reflected light beam 139 is focused by the focusing lens 151 and heads toward the unpolarized beam splitter 154. The unpolarized beam splitter has the function of reflecting and transmitting light incident on the separation surface at a predetermined ratio regardless of the polarization direction. In this embodiment, the unpolarized beam splitter 154 reflects and transmits incident light at a ratio of 50:50. The intensity distribution of the cross-sections of the first reflected light beam 140a that has passed through the unpolarized beam splitter 154 and the second reflected light beam 140b that has been reflected are symmetrical to each other.

[0078] The first reflected light beam 140a, having passed through the unpolarized beam splitter 154, is focused and incident on the first cylindrical lens 155a. The first cylindrical lens 155a is positioned with its cylindrical axis tilted 45 degrees with respect to the Y axis in the XY plane. The first reflected light beam 140a, having passed through the first cylindrical lens 155a, is given astigmatism and incident on the first photodetector 156a. The first photodetector 156a is a four-segment photodiode, and its segmentation lines are positioned along the X and Y axes. A first focus error signal (FES1) is generated from the output signals of each element (A1, B1, C1, and D1) of the first photodetector 156a using equation 3.

[0079]

number

[0080] Meanwhile, the second reflected light beam 140b reflected by the unpolarized beam splitter 154 is focused and incident on the second cylindrical lens 155b. The second cylindrical lens 155b is positioned with its cylindrical axis tilted 45 degrees with respect to the Y axis in the YZ plane. The second reflected light beam 140b is transmitted through the second cylindrical lens 155b, resulting in astigmatism, and then incident on the second photodetector 156b. The second photodetector 156b is a four-segment photodiode, and its segmentation lines are positioned along the Y and Z axes. A second focus error signal (FES2) is generated from the output signals of each element (A2, B2, C2, and D2) of the second photodetector 156b using Equation 4.

[0081]

number

[0082] In this embodiment 6, the focus error signal FES is calculated as the average value of the first focus error signal FES1 and the second focus error signal FES2. That is, it is calculated using Equation 5.

[0083]

number

[0084] With the above configuration, if minute irregularities on the focus target surface cause distortion of the light spot shape on the photodetector surface, the components of the distortion of the light spot shape in the first photodetector 156a and the second photodetector 156b are symmetrical with respect to each other. Therefore, by calculating the average value of the focus error signals FES1 and FES2 generated from the first photodetector 156a and the second photodetector 156b, the fluctuating component of the focus error signal due to the distortion of the light spot shape is canceled out. Accordingly, according to this embodiment, it is possible to obtain a stable focus error signal even if there are minute irregularities on the focus target surface.

[0085] <Regarding variations of the present invention> The present invention is not limited to the embodiments described above, and various modifications are included. For example, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment without departing from the spirit of the invention, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0086] The microscope according to the present invention is not limited to a transmission bright-field microscope, but may also be a fluorescence microscope, for example. The illumination method may be reflected light. The specimen to be imaged is not limited to tissue sections. Each embodiment of the present invention can also be applied to glass slide specimens using blood or body fluids. The wavelength of the laser light of the focus detection optical system is not limited to 785 nm. It is preferable to select a wavelength outside the wavelength range (visible range) of the microscope's illumination light source. The illumination light source of the microscope does not have to be an LED. It may be a tungsten lamp, halogen lamp, xenon lamp, etc.

[0087] As described in the embodiments above, the present invention combines real-time focus detection using the astigmatism method with correction of the focus error signal using a map. Compared to conventional methods that control the position of the objective lens using only a focus map, the present invention has the advantage of not being affected by temperature drift of the sample height that occurs after map creation.

[0088] In the embodiments described above, the arithmetic unit 200 can be configured by hardware such as a circuit device that implements its functions, or by a computing device such as a CPU (Central Processing Unit) executing software that implements its functions. [Explanation of Symbols]

[0089] 1. Microscope Optical System 2. Focus detection optical system 3 Illumination optical system 101 Light-emitting diodes 102 Collector lens 103 Condenser Lens 104 Field of View Aperture 105 Aperture diaphragm 111 Microscope slides 112 Cover glass 121 Objective lens 122 Objective lens position adjustment mechanism 123 Imaging lens 124 image sensors 131 Laser Diode 132 Collimator lens 133 Collimator lens position adjustment mechanism 134 Aperture 135 Focus detection beam 136 1 / 2 wavelength plate 137 Polarizing Beam Splitter 138 1 / 4 wave plate 139 Reflected light beam 140a First reflected light beam 140b Second reflected beam 141 Dichroic Filter 151 Focusing lens 152 Cylindrical Lens 153 Photodetector 154 Non-polarized beam splitter 155a First Cylindrical Lens 155b Second Cylindrical Lens 156a First photodetector 156b Second photodetector 200 Arithmetic section 301 Tissue section 302 Mounting medium 401 Focus Learning Points

Claims

1. A microscope for observing a sample covered with a cover slip, A collimator lens that makes the light emitted from the light source into nearly parallel light. An objective lens that focuses the light transmitted through the collimator lens onto the sample, A position adjustment mechanism for moving the position of the collimator lens, A detector for detecting reflected light from the aforementioned sample, Equipped with, The position adjustment mechanism moves the position of the collimator lens so that, when the focus of the objective lens coincides with the object being observed in the sample, the light that has passed through the objective lens is focused onto the surface of the cover glass. The detector detects the reflected light as a focus error signal representing the amount of deviation of the focal point of the light that has passed through the objective lens from the focusing surface. A microscope characterized by the following features.

2. The position adjustment mechanism moves the position of the collimator lens so that, when the objective lens is pre-adjusted to focus on the sample, the light that has passed through the objective lens is focused onto the surface of the cover glass. The microscope according to claim 1, characterized in that it is a feature of the present invention.

3. The position adjustment mechanism moves the position of the collimator lens in a direction that brings the focus error signal closer to zero. The microscope according to claim 1, characterized in that it is a feature of the present invention.

4. The microscope further includes a calculation unit that acquires a correction value map describing the correction values ​​used to correct the fluctuations in the focus error signal caused by the thickness of the cover glass for each planar position on the surface of the cover glass, The calculation unit corrects the focus error signal using the correction value at each of the planar positions described in the correction value map. The calculation unit controls the position of the objective lens in a direction that brings the corrected focus error signal closer to zero. The microscope according to claim 1, characterized in that it is a feature of the present invention.

5. The position adjustment mechanism moves the collimator lens to a position where the focus error signal becomes zero. The calculation unit adjusts the focus of the objective lens to match the sample for each planar position and then acquires the focus error signal. The calculation unit records the focus error signal acquired for each planar position as the correction value. The microscope according to feature 4.

6. The microscope further includes a calculation unit that acquires a position map describing the target position of the collimator lens where the focus error signal becomes zero, for each planar position on the surface of the cover glass. The calculation unit controls the position of the collimator lens to the target position at each of the planar positions described in the position map. The microscope according to claim 1, characterized in that it is a feature of the present invention.

7. The position adjustment mechanism moves the collimator lens to a position where the focus error signal becomes zero. The calculation unit adjusts the focus of the objective lens to match the sample for each planar position, then moves the collimator lens to a position where the focus error signal becomes zero, and the calculation unit records the position of the collimator lens where the focus error signal becomes zero for each planar position as the target position. The microscope according to claim 6, characterized in that it is a microscope.

8. The microscope further includes a map creation device that creates a correction value map describing the correction values ​​used to correct the fluctuations in the focus error signal caused by the thickness of the cover glass for each planar position on the surface of the cover glass. The microscope further includes a calculation unit that corrects the focus error signal using the correction value map, The calculation unit corrects the focus error signal using the correction value at each of the planar positions described in the correction value map. The calculation unit controls the position of the objective lens in a direction that brings the corrected focus error signal closer to zero. The microscope according to feature 4.

9. The microscope further includes a map creation device that creates a position map describing the target position of the collimator lens where the focus error signal is zero, for each planar position on the surface of the cover glass. The microscope further includes a calculation unit that controls the position of the collimator lens using the position map, The calculation unit controls the position of the collimator lens to the target position at each of the planar positions described in the position map. The microscope according to claim 6, characterized in that it is a microscope.

10. The microscope further includes light-dividing elements that guide the reflected light to a first detection optical system and a second detection optical system, respectively. The light splitting element guides the reflected light that has passed through the light splitting element to the first detection optical system, and guides the reflected light that has been reflected from the light splitting element to the second detection optical system. The intensity distribution of the reflected light directed to the first detection optical system and the intensity distribution of the reflected light directed to the second detection optical system are inverted symmetrically from each other. The microscope according to claim 1, characterized in that it is a feature of the present invention.

11. The detector detects the focus error signal based on the astigmatism method. The microscope according to claim 1, characterized in that it is a feature of the present invention.

12. The detector comprises at least four or more detection elements, Each of the detection elements detects the reflected light and outputs the detection result. The microscope according to claim 1, characterized in that it is a feature of the present invention.

13. An observation method for observing a sample covered with a cover slip, A step of making the light emitted from the light source into nearly parallel light using a collimator lens. The steps of focusing the light transmitted through the collimator lens onto the sample using an objective lens, A step of moving the position of the collimator lens, A step of detecting reflected light from the sample, It has, In the step of moving the position, the position of the collimator lens is moved such that the light passing through the objective lens is focused onto the surface of the cover glass when the focal point of the objective lens coincides with the object of observation of the sample. In the detection step described above, the reflected light is detected as a focus error signal representing the amount of deviation of the focal point of the light that has passed through the objective lens from the plane of focus. An observation method characterized by the following.

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