Microscope and observation method

Abstract

The purpose of the present invention is to provide a microscope with an autofocus mechanism that achieves both high-speed focus tracking and focus accuracy. In a microscope according to the present invention, the position of a collimator lens is moved so that light that has passed through an objective lens is focused on the surface of a cover glass, and a reflected light from a sample is detected as a focus error signal indicating the amount of deviation of the focal point of the light that has passed through the objective lens from the surface to be focused (see fig. 1).

Description

Microscope, observation method

[0001] The present invention relates to a microscope.

[0002] Pathology is a field of medicine that diagnoses diseases by examining tissues, surgically removed organs, and body fluids under a microscope. In recent years, the widespread use of digital imaging, particularly the development and widespread use of WSI (whole slide imaging), has led to the evolution of traditional pathology into digital pathology. WSI is a system in which an entire glass slide specimen is imaged in advance using a high-magnification microscope, which is then imported into a computer as a digital image, which is then displayed and observed on a monitor. The digital image capturing device is called a WSI scanner.

[0003] In many digital pathology applications, the specimen scanning process is a major bottleneck in the workflow, and there is a demand for improved throughput (shortened scanning time) in the scanning process.

[0004] Meanwhile, in the microscopes of WSI scanners, 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. Autofocus methods for WSI scanners are broadly divided into two types based on the focus detection method: reflective and image-based. The reflective type uses a dedicated light source to receive light reflected from the sample and detects the height of a 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] The astigmatism method, a type of reflective focus detection method, detects defocus by irradiating a focused laser beam onto a 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 fact that the shape of the light spot on the detector changes depending on the amount of defocus. Because the astigmatism method uses a light source and detection system dedicated to focus detection, it does not require the processing of captured images that is required in image-based focus detection methods. Furthermore, since it can be implemented with simple signal processing, it excels in high-speed focus tracking and is an effective method for reducing the scanning 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 a microscope apparatus capable of shortening the time required to focus an objective lens on an object. The document describes a technology in which "the autofocus device 20 includes a drive unit 25 that moves the objective lens 11 in the optical axis direction, a detection unit 28 that detects whether an object is placed at a detection position DP that is shifted 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 the object is placed at the detection position DP, drives the drive unit 25 to move the objective lens 11 by the reference distance, thereby controlling the objective lens 11 to focus on the object" (see abstract).

[0007] Japanese Patent Application Laid-Open No. 2022-137838

[0008] One example of a glass slide specimen, which serves as an imaging target for a WSI scanner, is a tissue section mounted on a glass slide and mounted with a cover glass using a mounting medium. To ensure that the focus detection method based on the astigmatism method functions ideally, i.e., with high focus detection sensitivity, the focus detection laser beam (hereinafter referred to as the focus detection beam) must be focused and irradiated onto the focus target surface. When the astigmatism method is applied to autofocusing a glass slide specimen, the specimen is irradiated with laser light using the objective lens of a microscope, and the focus target surface is the main reflecting surface of the irradiated light, i.e., the surface of the cover glass.

[0009] However, when imaging a tissue section, the position (height) of the objective lens must be adjusted so that the tissue section is in focus. This constraint prevents the focus detection beam from being focused on the surface of the cover glass, which is the target surface. In many cases, the focus detection beam is a parallel beam, and the focal point of the objective lens is the tissue section. Therefore, the beam is spread on the cover glass surface, resulting in a significant decrease in focus detection sensitivity, i.e., the change in focus error signal relative to the amount of defocus, compared to the ideal case. This decrease in focus detection sensitivity leads to a decrease in focus accuracy.

[0010] SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems and to provide a microscope with an autofocus mechanism that achieves both high speed focus tracking and high focus accuracy.

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

[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 objects, configurations, advantages, etc. of the present invention will become clear from the description of the following embodiments.

[0013] 6 shows an example of the configuration of an imaging optical system provided in a microscope according to embodiment 1. FIG. 7 is a diagram illustrating the principle of detecting a focus error, i.e., the amount of deviation of the focal point of an illumination light beam from a focus target surface, using an astigmatism method. FIG. 8 is a diagram schematically illustrating one form of a focused state of a focus detection beam 139. FIG. 9 is a diagram illustrating the shape of a light spot on a photodetector 153 when the position of the objective lens 121 is changed based on the state of FIG. 3 , where defocus=0. FIG. 10 is a graph plotting the relationship between defocus and a focus error signal (FES) when the state of FIG. 3 is set to defocus=0. FIG. 11 is a diagram schematically illustrating another form of a focused state of a focus detection beam 139. FIG. 12 is a diagram illustrating the shape of a light spot on a photodetector 153 when the position of the objective lens 121 is changed based on the state of FIG. 10 , where defocus=0. FIG. 13 is a graph plotting the relationship between defocus and a focus error signal (FES) when the state of FIG. 11 is set to defocus=0. FIG. 14 is a diagram illustrating the focused state of a focus detection beam 139. 10 is a diagram showing the shape of a light spot on the photodetector 153 when the position of the objective lens 121 is changed based on the state of FIG. 9 where defocus = 0. FIG. 11 is a graph plotting the relationship between defocus and focus error signal (FES) when the state of FIG. 9 is set to defocus = 0. FIG. 12 is a flowchart explaining a procedure for performing autofocus control by adjusting the position of the collimator lens 132 to match an arbitrary glass slide specimen. FIG. 13 is a conceptual diagram showing the principle of how focus errors occur due to uneven cover glass thickness. FIG. 14 is a top view showing an example of the arrangement of focus learning points. FIG. 15 is a flowchart explaining a procedure for creating a focus error signal correction value map and performing autofocus control using a focus error signal corrected by the data. FIG. 16 is a flowchart explaining a procedure for creating a collimator lens position map and performing autofocus control while dynamically controlling the position of the collimator lens 132 by the data. FIG. 17 is a diagram showing an example of the configuration of an imaging optical system provided in a microscope according to a sixth embodiment.

[0014] <Embodiment 1: Basic Configuration (Beam Focus Point Position Adjustment)> <Configuration of Overall Optical System> Fig. 1 shows an example configuration of an imaging optical system provided in a 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 a calculation unit 200. The coordinate axes are defined as follows: the optical axis direction of the microscope optical system 1 is the Z axis; the direction parallel to the paper surface among the in-plane directions of a glass slide specimen (described later) is the X axis; and the direction perpendicular to the paper surface is the Y axis. The calculation unit 200 controls each unit of the microscope and executes the flowcharts described later.

[0015] <Configuration of Microscope> The configuration of the microscope optical system 1 is classified as that of a transmission type 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 forms a real image of the light source on the condenser lens 103, and the condenser lens 103 forms 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 Kohler illumination, which uniformly illuminates the sample surface. The illumination optical system 3 also comprises a field stop 104 and an aperture stop 105. The field stop 104 adjusts the illumination range on the sample surface, and the aperture stop 105 adjusts the numerical aperture of the illumination light.

[0016] The glass slide specimen, which is the object to be imaged, is a tissue slice placed on a glass slide and sealed with a cover glass 112 using a mounting agent.

[0017] The objective lens 121 and the imaging lens 123 constitute an infinity corrected optical system, which forms an image of the tissue slice in the glass slide specimen on the sensor surface of the imaging element 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 focus errors based on the astigmatism method. A laser diode 131, which serves as a light source, emits linearly polarized infrared laser light with a wavelength of 785 nm. The emitted laser light is collimated by a collimator lens 132 to form a substantially parallel beam, and then the beam diameter is limited to a predetermined size by an aperture 134 to form a focus detection beam 135. The focus detection beam 135 passes through a λ / 2 plate 136 and enters a polarizing beam splitter 137.

[0019] The polarizing beam splitter 137 has the function of transmitting almost 100% of p-polarized light (polarized light whose electric field component is parallel to the plane of incidence) incident on the separation surface and reflecting almost 100% of s-polarized light (polarized light whose electric field component is perpendicular to the plane of incidence). By adjusting the rotation angle of the λ / 2 plate 136 about the optical axis with respect to the focus detection beam 135, the polarization direction of the light passing through the λ / 2 plate 136 can be arbitrarily changed, and the intensity ratio between the transmitted light and the 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 passing through the λ / 2 plate 136 is p-polarized, and almost 100% of the focus detection beam 135 is transmitted through the polarizing beam splitter 137.

[0020] The focus detection beam 135, which is transmitted through the polarizing beam splitter 137 at almost 100%, is converted into circularly polarized light by passing through a λ / 4 plate 138, which is positioned with its fast axis tilted 45 degrees with respect 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 into the optical path of the microscope optical system 1 by a dichroic filter 141, which is positioned in the optical path of the microscope optical system 1. The focus detection beam 135 is then focused by the objective lens 121 and irradiated onto a glass slide specimen, which is the object to be imaged.

[0021] The beam reflected by the surface of the cover glass 112 of the glass slide specimen (hereinafter referred to as the reflected light beam 139 or the focus detection beam 139) is converted back into a substantially parallel beam by the objective lens 121 and then converted back into linearly polarized light by the λ / 4 plate 138. At this time, the direction of rotation of the circularly polarized light is reversed by reflection on the surface of the cover glass 112, so that the direction of the linearly polarized light becomes s-polarized light, rotated 90 degrees from the light on the outbound path. 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 toward the focusing lens 151.

[0022] The reflected light beam 139 is focused by a focusing lens 151 and then enters a cylindrical lens 152. The cylindrical lens 152 is disposed so that its cylindrical axis is tilted 45 degrees with respect to the Y axis in the XY plane. The reflected light beam 139 is given astigmatism by passing through the cylindrical lens 152 and then enters a photodetector 153. The photodetector 153 is a four-segment photodiode, and is disposed so that its dividing lines are aligned with the X axis and the Y axis. A focus error signal (FES), which will be 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 by Astigmatism Method> FIG. 2 is a diagram showing the principle of detecting a focus error, that is, the amount of deviation of the focal point of an illumination light beam from a focus target surface, using the astigmatism method.

[0024] The reflected light beam 139 is converged by the focusing lens 151, while being subjected to astigmatism by the cylindrical lens 152. The cylindrical lens 152 acts as a lens only in a single direction, and in the direction perpendicular to that, it acts like a parallel plate and does not act as a lens. 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 as seen from a 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 as seen from a cross section where the lens effect is present does not form a focus, and a thin line image (focal line) is obtained.

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

[0026]

[0027] When measuring defocus using the astigmatism method, the center of the photodetector 153 is aligned with the position where the reflected light beam from the focal plane of the objective lens 121 becomes circular, as shown in Figure 2 (2). The photodetector 153 is composed of a four-segment photodiode, 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]

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

[0030] The sign of the FES depends on the installation angle of the cylindrical lens 152 with the optical axis direction as the center of rotation. In this embodiment, the cylindrical lens 152 is installed at a 45-degree angle with the optical axis direction as the center of rotation. The optical axis direction here 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 is a diagram schematically showing one form of the focused state of the focus detection beam 139. The mounting medium 302 is used to mount the tissue slice 301. In this state, 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 on the surface of the cover glass 112. In other words, this is one form 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 taken as the defocus = 0 as the reference. Figures 4 (1), (2), and (3) show the cases when the defocus is -20, 0, and +20 μm, respectively. The size of the photodetector is 0.5 mm x 0.5 mm. The plot range is the same as the size of the photodetector, 0.5 mm x 0.5 mm. In this way, the spot shape on the photodetector 153 changes to become elongated in the diagonal direction as the defocus changes.

[0033] 5 is a graph plotting the relationship between defocus and the focus error signal (FES) when the state of FIG. 3 is set to defocus=0. As can be seen, the focus error signal varies linearly with defocus between positive and negative peaks, indicating that the astigmatism method is functioning ideally.

[0034] However, in the state shown in FIG. 3, the focus of the objective lens 121 is 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 slice 301, cannot be achieved.

[0035] 6 is a diagram schematically illustrating another form of the focused state of the focus-detecting beam 139. The focus-detecting beam 139 is a parallel beam, as in the case of FIG. 3. Meanwhile, the position of the objective lens 121 is adjusted so as to focus on the tissue slice 301. In other words, this shows the actual operating state of the WSI scanner. However, in this state, the focus-detecting beam 139 is focused on the tissue slice 301 and spreads on the surface of the cover glass 112, which is the focus target surface.

[0036] FIG. 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 FIG. 6 being assumed to be defocus = 0 as a reference. Figures 7(1), (2), and (3) show the cases where the defocus is -20, 0, and +20 μm, respectively. The plot range is 2.0 mm x 2.0 mm, unlike FIG. 3. The size of the photodetector (0.5 mm x 0.5 mm) is indicated by a white dashed line in the figure. As such, in the state in FIG. 6, the light spot on the photodetector 153 expands significantly and extends beyond the photodetector 153. As a result, the focus error signal hardly changes with changes in defocus, making defocus detection difficult. Even if a photodetector large enough to receive the entire light spot is used, the change in the light spot shape with changes in defocus is significantly smaller than the ideal state in FIG. 3.

[0037] 8 is a graph plotting the relationship between defocus and the focus error signal (FES) when the state of FIG. 6 is set to defocus = 0. As shown, the focus error signal hardly changes near defocus = 0, meaning that the focus detection sensitivity is extremely low.

[0038] 9 is a diagram schematically illustrating the focusing state of the focus detection beam 139 in this embodiment. The position of the objective lens 121 is adjusted so as to focus on the tissue slice 301. In other words, this is the actual operating state of the WSI scanner. However, unlike the case of FIG. 3, the focus detection beam 139 is weakly focused when it enters the objective lens 121 and focuses on the surface of the cover glass 112, which is the focus target surface. This configuration enables the objective lens 121 to be positioned at a position suitable for imaging, while allowing focus detection using the astigmatism method to function ideally.

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

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

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

[0042] 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 beam traveling direction. As a result, the laser light transmitted through the collimator lens 132 becomes a weakly focused beam that travels. The weakly focused focus detection beam 135 is focused by the objective lens 121, and the position of the focused point is located in front of the focal plane (focal length) of the objective lens 121.

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

[0044] <Embodiment 2: Method for Adjusting Beam Focus Point Position> <Adjustment to Bring the Focus Error Signal Closer to Zero> 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 the astigmatism method. The microscope has the same configuration as in Embodiment 1. First, a procedure for setting the focus detection optical system 2 will be described. The photodetector 153 in the focus detection optical system 2 in FIG. 1 is positioned so that the focus error signal becomes zero when the focus detection beam 135 is exactly focused on the surface of the cover glass 112. An example of a procedure for positioning the photodetector 153 at such a position is shown below.

[0045] First, the position of the objective lens 121 is adjusted so that the focal plane of the objective lens 121 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 an image captured by the microscope optical system 1 so that minute scratches on the surface of the cover glass 112 and minute dust particles adhering to the surface can be clearly seen. Next, the photodetector 153 is positioned at a position where the focus error signal becomes zero when the focus detection beam 135 is a parallel beam. In other words, the photodetector 153 is positioned at a position where the reflected light beam 139 becomes circular. Through the above procedure, 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 exactly focused on the surface of the cover glass 112.

[0046] 12 is a flowchart explaining the procedure for performing autofocus control by adjusting the position of the collimator lens 132 to match an arbitrary glass slide specimen. The focus detection optical system 2 is assumed to be preset at a position where the focus error signal becomes zero when the focus detection beam 135 is exactly focused on the surface of the cover glass 112. This flowchart (and the following flowcharts as well) is executed by the calculation unit 200. Each step in FIG. 12 will be explained below.

[0047] Step S12: At an arbitrary representative point (XY coordinates) on the glass slide specimen, preferably at a location where the tissue slice to be imaged is present, the objective lens 121 is focused on the tissue slice. That is, the objective lens 121 is moved to a position where the image of the tissue slice is most clearly captured.

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

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

[0050] The above series of procedures is performed every time the glass slide specimen is replaced. This allows the focus detection beam 135 to always be focused on the surface of the cover glass 112, even if the height of the cover glass surface changes due to variations in the thickness of the glass slide and cover glass in each glass slide specimen, ensuring high focus detection sensitivity.

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

[0052] <Focus Error Due to Uneven Thickness of Cover Glass> Figure 13 is a conceptual diagram illustrating the principle of how focus error occurs due to uneven thickness of the cover glass. Figure 13 corresponds to a side cross-sectional view of the cover glass. Autofocus control controls the position of the objective lens 121 so as to follow changes in the height of the surface of the cover glass 112. However, if the thickness of the cover glass 112 varies depending on the location to be imaged (XY coordinates), the distance from the surface of the cover glass 112 to the tissue slice 301 to be imaged changes, and this change results in a focus error.

[0053] <Map Creation and Focus Learning Point Arrangement> Figure 14 is a top view showing an example of the arrangement of focus learning points. An imaging range is set to include the entire tissue slice 301 to be imaged, and focus learning points 401 are arranged at a predetermined interval within the imaging range. The arrangement interval of focus learning points 401 is preferably less than 1 / 2 the reciprocal of the maximum spatial frequency (minimum spatial period) of cover glass thickness variation.

[0054] <Interpolation> The correction values ​​used to correct the focus error signal can be set for each X and Y coordinate on the surface of the tissue slice 301. Data describing these correction values ​​is referred to as a focus error signal correction value map. One form of the focus error signal correction value map is a lookup table format in which the correspondence between X and Y coordinates and focus error signal correction values ​​is stored as an array. Focus error signal correction values ​​at X and Y coordinates between focus learning points not included in the map are calculated by interpolation using data from multiple nearby focus learning points. Alternatively, a mathematical formula for calculating the focus error signal correction value as a function of the X and Y coordinates may be used.

[0055] 15 is a flowchart illustrating a procedure for creating a focus error signal correction value map and performing autofocus control using a focus error signal corrected by that data. Each step in FIG. 15 will be described below.

[0056] Step S22: At an arbitrary representative point (XY coordinates) on the glass slide specimen, preferably at a location where the tissue slice to be imaged is present, the objective lens 121 is focused on the tissue slice. That is, the objective lens 121 is moved to a position where the image of the tissue slice is most clearly captured.

[0057] Step S23: The collimator lens 132 is moved to a position where the focus error signal becomes zero by the collimator lens position adjustment mechanism 133. By this step, the focus detection beam 135 is focused on the surface of the cover glass 112 at the 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 slice. That is, the objective lens 121 is moved to a position where the captured image of the tissue slice becomes the clearest.

[0060] Step S26: A focus error signal is detected and recorded as a 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 step S28. If NO, return to step S24.

[0062] Step S28: A focus error signal correction value map is created, which indicates the relationship between X and Y coordinates and the focus error signal correction value at those coordinates.

[0063] Step S29: This is an autofocus control step in which a focus error signal is detected, and the focus error signal is corrected by subtracting a focus error signal correction value at the current X and Y coordinates from the detected focus error signal, and feedback control is repeatedly performed to move the objective lens 121 in a direction in which the corrected focus error signal approaches zero.

[0064] <Fourth Embodiment: Alternative Map> <Flowchart> In a fourth embodiment of the present invention, a collimator lens position map will be described which is created from a different perspective from the focus error signal correction value map described in the third embodiment. The configuration of the microscope is the same as that of the first embodiment.

[0065] 16 is a flowchart illustrating a procedure for creating a collimator lens position map and performing autofocus control while dynamically controlling the position of the collimator lens 132 based on the data. Each step in FIG. 16 will be described below.

[0066] Step S32: At an arbitrary representative point (XY coordinates) on the glass slide specimen, preferably at a location where the tissue slice to be imaged is present, the objective lens 121 is focused on the tissue slice. That is, the objective lens 121 is moved to a position where the image of the tissue slice is most clearly captured.

[0067] Step S33: The collimator lens 132 is moved to a position where the focus error signal becomes zero by the collimator lens position adjusting mechanism 133. By this step, the focus detection beam 135 is focused on the surface of the cover glass 112 at the 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 slice. That is, the objective lens 121 is moved to a position where the captured image of the tissue slice becomes the clearest.

[0070] Step S36: The collimator lens 132 is moved to a position where the focus error signal becomes zero, and this position is recorded 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 step S38. If NO, return to step S34.

[0072] Step S38: A collimator lens position target value map is created, which indicates the relationship between XY coordinates and the collimator lens position target values ​​at those coordinates.

[0073] Step S39: This is an autofocus control step. The position of the objective lens 121 is controlled in the same manner 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 in which the focus error signal approaches zero.

[0074] In the fourth embodiment, the position of the collimator lens 132 is feedforward controlled to a target position according to the X and Y coordinates. This target position is extracted from a 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 for 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 a separate device (map creation device) having a configuration equivalent to that of the microscopes of the above embodiments. For example, the map creation device can create a map of the next slide to be observed by the microscope in advance, thereby enabling the observation process to be carried out smoothly. The map creation device does not necessarily have to have exactly the same configuration as the microscope of the present invention, as long as it has a configuration that can create the maps described in embodiments 3 and 4.

[0076] <Sixth embodiment: Dual astigmatism method> Fig. 17 is a diagram showing an example of the configuration of an imaging optical system provided in a microscope according to a sixth embodiment of the present invention. The sixth embodiment relates to a different configuration of the focus detection optical system. The other configurations are the same as those of the above embodiments.

[0077] The s-polarized reflected light beam 139 that has passed through the λ / 4 plate 138 is reflected at 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 non-polarizing beam splitter 154. The non-polarizing beam splitter has the function of reflecting and transmitting light incident on its splitting surface at a predetermined ratio regardless of the polarization direction. The non-polarizing beam splitter 154 of this embodiment reflects and transmits incident light at a 50:50 ratio. The cross-sectional intensity distributions of the first reflected light beam 140a and the second reflected light beam 140b that have passed through the non-polarizing beam splitter 154 are linearly symmetrical to each other.

[0078] The first reflected light beam 140a transmitted through the non-polarizing beam splitter 154 is converged and incident on a first cylindrical lens 155a. The first cylindrical lens 155a is disposed with its cylindrical axis tilted at 45 degrees with respect to the Y axis in the XY plane. The first reflected light beam 140a is given astigmatism by transmitting through the first cylindrical lens 155a and then incident on a first photodetector 156a. The first photodetector 156a is a four-segment photodiode, and is disposed so that its division lines are aligned with the X axis and the Y axis. Each element (A) of the first photodetector 156a １ , B １ , C １ , and D １ ) output signal, the first focus error signal (FES) is calculated using Equation 3. １ ) is generated.

[0079]

[0080] On the other hand, the second reflected light beam 140b reflected by the non-polarizing beam splitter 154 is converged and enters a second cylindrical lens 155b. The second cylindrical lens 155b is disposed with its cylindrical axis tilted at 45 degrees with respect to the Y axis in the YZ plane. The second reflected light beam 140b is given astigmatism by passing through the second cylindrical lens 155b and then enters a second photodetector 156b. The second photodetector 156b is a four-segment photodiode, and is disposed so that its division lines are aligned with the Y axis and the Z axis. Each element (A) of the second photodetector 156b２ , B ２ , C ２ , and D ２ ) output signal, a second focus error signal (FES) is calculated using Equation 4. ２ ) is generated.

[0081]

[0082] The focus error signal FES in the sixth embodiment is a first focus error signal FES １ and the second focus error signal FES ２ That is, it is calculated using Equation 5.

[0083]

[0084] With the above configuration, when the shape of the light spot on the photodetector surface is disturbed due to minute irregularities on the focus target surface, the components of the disturbance in the shape of the light spot on the first photodetector 156a and the second photodetector 156b are in a line-symmetric relationship with each other. Therefore, the focus error signal FES generated by the first photodetector 156a and the second photodetector 156b １ and FES ２ By calculating the average value of the above, the fluctuation component of the focus error signal due to the disturbance of the light spot shape is cancelled. Therefore, 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] <Modifications of the present invention> The present invention is not limited to the above-described embodiment, and includes various modifications. For example, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, or to add the configuration of another embodiment to the configuration of one embodiment, without departing from the spirit of the invention. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with another configuration.

[0086] The microscope according to the present invention is not limited to a transmission bright-field microscope, but may be, for example, a fluorescence microscope. The illumination method may be epi-illumination. The specimen to be imaged is not limited to a tissue section. Each embodiment of the present invention can also be applied to a glass slide specimen using blood or body fluid. 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 range outside the wavelength range (visible range) of the illumination light source of the microscope. The illumination light source of the microscope does not have to be an LED. A tungsten lamp, a halogen lamp, a xenon lamp, etc. may also be used.

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

[0088] In the above embodiments, the calculation unit 200 can be configured by hardware such as a circuit device that implements its functions, or can be configured by a calculation device such as a CPU (Central Processing Unit) that executes software that implements its functions.

[0089] 1 Microscope optical system 2 Focus detection optical system 3 Illumination optical system 101 Light-emitting diode 102 Collector lens 103 Condenser lens 104 Field stop 105 Aperture stop 111 Slide glass 112 Cover glass 121 Objective lens 122 Objective lens position adjustment mechanism 123 Imaging lens 124 Image sensor 131 Laser diode 132 Collimator lens 133 Collimator lens position adjustment mechanism 134 Aperture 135 Focus detection beam 136 Half-wave plate 137 Polarizing beam splitter 138 Quarter-wave plate 139 Reflected light beam 140a First reflected light beam 140b Second reflected light beam 141 Dichroic filter 151 Focusing lens 152 Cylindrical lens 153 Photodetector 154 Non-polarizing beam splitter 155a First cylindrical lens 155b Second cylindrical lens 156a First photodetector 156b Second photodetector 200 Calculation unit 301 Tissue slice 302 Mounting medium 401 Focus learning point

Claims

1. A microscope for observing a sample covered by a cover glass, comprising: a collimator lens that converts light emitted from a light source into approximately parallel light; an objective lens that focuses the light that has passed through the collimator lens onto the sample; a position adjustment mechanism that moves the position of the collimator lens; and a detector that detects reflected light from the sample, wherein the position adjustment mechanism 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 the detector detects the reflected light as a focus error signal that indicates the amount of deviation of the focal point of the light that has passed through the objective lens from a focus target surface.

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

3. The microscope according to claim 1, wherein said position adjustment mechanism moves the position of said collimator lens in a direction in which said focus error signal approaches zero.

4. The microscope according to claim 1, further comprising a calculation unit that acquires a correction value map that describes, for each planar position on the surface of the cover glass, correction values ​​used to correct fluctuations in the focus error signal caused by the thickness of the cover glass, the calculation unit corrects the focus error signal using the correction values ​​at each of the planar positions described in the correction value map, and the calculation unit controls the position of the objective lens in a direction in which the corrected focus error signal approaches zero.

5. The microscope according to claim 4, characterized in that: the position adjustment mechanism moves the collimator lens to a position where the focus error signal is zero; the calculation unit acquires the focus error signal after adjusting the focus of the objective lens to the sample for each planar position; and the calculation unit records the focus error signal acquired for each planar position as the correction value.

6. The microscope according to claim 1, further comprising a calculation unit that acquires a position map that describes a target position of the collimator lens at which the focus error signal is zero for each planar position on the surface of the cover glass, and 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.

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

8. The microscope according to claim 4, further comprising a map creation device that creates a correction value map in which correction values ​​used to correct fluctuations in the focus error signal due to the thickness of the cover glass are described for each planar position on the surface of the cover glass, the microscope further comprising 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, and the calculation unit controls the position of the objective lens in a direction in which the corrected focus error signal approaches zero.

9. The microscope according to claim 6, further comprising a map creating device that creates a position map that describes, for each planar position on the surface of the cover glass, a target position of the collimator lens where the focus error signal is zero; the microscope further comprises a calculation unit that controls the position of the collimator lens using the position map; and 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.

10. The microscope according to claim 1, further comprising a light dividing element which respectively guides the reflected light to a first detection optical system and a second detection optical system, the light dividing element directing the reflected light which has passed through the light dividing element to the first detection optical system and directing the reflected light reflected from the light dividing element to the second detection optical system, and the intensity distribution of the reflected light guided to the first detection optical system and the intensity distribution of the reflected light guided to the second detection optical system are linearly symmetrical inverses of each other.

11. The microscope according to claim 1, wherein the detector detects the focus error signal based on an astigmatism method.

12. The microscope according to claim 1, characterized in that the detector is provided with at least four or more divided detection elements, each of which detects the reflected light and outputs the detection result.

13. An observation method for observing a sample covered with a cover glass, comprising: a step of converting light emitted from a light source into approximately parallel light using a collimator lens; a step of focusing the light that has passed through the collimator lens onto the sample using an objective lens; a step of moving the position of the collimator lens; and a step of detecting reflected light from the sample, wherein in the step of moving the position, the position of the collimator lens is moved so that the light that has passed through the objective lens is focused on the surface of the cover glass, and in the step of detecting, the reflected light is detected as a focus error signal that indicates the amount of deviation from a focus target surface of the focal point of the light that has passed through the objective lens.

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