WO2022162791A1 - 校正用サンプル、校正用サンプルの製造方法及びオートフォーカス目標位置の校正方法 - Google Patents
校正用サンプル、校正用サンプルの製造方法及びオートフォーカス目標位置の校正方法 Download PDFInfo
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- WO2022162791A1 WO2022162791A1 PCT/JP2021/002860 JP2021002860W WO2022162791A1 WO 2022162791 A1 WO2022162791 A1 WO 2022162791A1 JP 2021002860 W JP2021002860 W JP 2021002860W WO 2022162791 A1 WO2022162791 A1 WO 2022162791A1
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Classifications
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
- G02B7/28—Systems for automatic generation of focusing signals
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
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- H04N23/60—Control of cameras or camera modules
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Definitions
- the present disclosure relates to a calibration sample, a calibration sample manufacturing method, and an autofocus target position calibration method.
- Autofocus is a function of moving, for example, the positions of an objective lens and a sample and fixing them at a position where the contrast of an object to be imaged is maximized.
- the image identification method is a method of calculating the contrast of the picked-up microscope image and fixing the position of the objective lens and the sample with the highest contrast.
- the optical method the bottom of the sample container is irradiated with light such as a laser from an optical system that is separate from the optical system that forms the microscope image, and the objective lens and sample are fixed at the position where the reflected light is most focused. be.
- the image identification method directly checks the contrast of the image captured by the optical microscope, so it is possible to capture the microscope image with the highest contrast.
- the position of the bottom of the sample container is detected by an optical system separate from the image captured by the optical microscope. can run.
- the optical method does not directly check the contrast of the image captured by the optical microscope. For this reason, the positions of the objective lens and the sample are fixed at the positions where the optical system different from the optical system that forms the microscope image is focused, and the calibration is performed to the position where the contrast of the image captured by the optical microscope is maximized. is done.
- Patent Document 1 ⁇ image data for observation is obtained by condensing light from a sample placed on a stage with an objective lens and capturing an image of the sample based on the condensed light.
- a generating microscope comprising an autofocus control for automatically focusing an image of a sample using autoexposure target values that depend on the type of sample and/or the mode of operation of the microscope. .” is stated.
- the target position of the feedback control of the objective lens performed by autofocus and the position where the maximum contrast of the object to be imaged can be obtained are performed so as to match. .
- the target position of the objective lens is changed.
- Calibration of the autofocus target position is executed, for example, each time an automatic imaging sequence such as time-lapse imaging is started.
- the position where the contrast is maximized can be set as the autofocus target position.
- Biological samples such as culture solutions contain solvents, and the imaging target may float in the solvents.
- the biological sample since the biological sample has a three-dimensional structure, necessary measurement information may not be obtained at the position where the contrast is maximized. Therefore, when the biological sample to be observed has a three-dimensional structure, the autofocus target position is set at a position where the observer can confirm the microscope image of the biological sample and obtain information on the floating observation target object. calibrated.
- FIG. 13 of Patent Document 1 describes a line-and-space sample in which a metal pattern is formed by vapor deposition or the like on the surface of a glass substrate.
- a sample without a three-dimensional structure (sufficiently thin) as in Patent Document 1 it is impossible to confirm how the actual biological sample looks. Therefore, it is preferable to reconfirm the appearance with an actual biological sample after calibration.
- the intensity of the reflected light in the optical autofocus changes, so the optical autofocus cannot be performed.
- sample containers for containing biological samples are often molded resin products, and have large variations in optical characteristics. For this reason, calibration using actual sample containers is preferred.
- the problem to be solved by the present disclosure is to provide a calibration sample that is also compatible with a three-dimensional structure and that does not easily change over time, a method for manufacturing a calibration sample, and a method for calibrating an autofocus target position.
- the calibration sample of the present disclosure is a calibration sample for an autofocus target position in an optical microscope, is arranged on the bottom side along the optical axis direction of the optical microscope, and is placed in the light-transmitting first resin.
- Light transmission containing a first layer in which a target object having contrast is arranged with respect to a first resin, and a second layer arranged to cover the first layer and composed of a second resin having a light transmission property. characterized by comprising a sample container made of a flexible resin.
- a calibration sample that is also compatible with a three-dimensional structure and that does not easily change over time, a method for manufacturing a calibration sample, and a method for calibrating an autofocus target position.
- FIG. 1 is a perspective view showing a calibration sample of the first embodiment
- FIG. FIG. 2 is a cross-sectional view showing the calibration sample of the first embodiment
- FIG. 4A is a process diagram showing the method of manufacturing the calibration sample according to the first embodiment, and shows a state in which the first resin in which the target fluid is dispersed is placed in the sample holder.
- FIG. 4 is a process diagram showing the method for manufacturing the calibration sample according to the first embodiment, and shows a first layer forming process
- FIG. 4 is a process chart showing the method for manufacturing the calibration sample of the first embodiment, and shows a second layer forming process.
- FIG. 4 is a diagram illustrating a method of calibrating an autofocus target position using a calibration sample according to the first embodiment
- FIG. 4A is a process diagram showing the method of manufacturing the calibration sample according to the first embodiment, and shows a state in which the first resin in which the target fluid is dispersed is placed in the sample holder.
- FIG. 4 is a process diagram showing the
- FIG. 4 is a schematic diagram of a microscope image in which the focus position is shifted from the position of the target object to the bottom side;
- FIG. 4 is a schematic diagram showing a state in which a microscope image is obtained in which the in-focus position matches the position of the target object and the contrast is maximized;
- FIG. 10 is a schematic diagram of a microscope image in which the focus position is shifted in a far direction when viewed from the bottom;
- FIG. 4 is a diagram showing a contrast graph of images obtained by scanning the objective lens in the optical axis direction a plurality of times, and a focus signal intensity graph used for feedback control of autofocus, with respect to the optical axis direction; be.
- FIG. 11 is a perspective view showing a calibration sample of the second embodiment;
- FIG. 11 is a perspective view showing a calibration sample of the second embodiment
- FIG. 5 is a cross-sectional view showing a calibration sample of the second embodiment
- FIG. 10A is a process diagram showing a method for manufacturing a calibration sample according to the second embodiment, and is a diagram showing a first layer forming step.
- FIG. 10 is a process diagram showing a method for manufacturing a calibration sample according to the second embodiment, and shows a second layer forming process;
- FIG. 10 is a diagram illustrating a method of calibrating an autofocus target position using a calibration sample according to the second embodiment;
- FIG. 4 is a schematic diagram of a microscope image in which the focus position is near the bottom surface of the first layer and the target object is out of focus; It is a schematic diagram of a microscope image when a focus position exists in the 1st layer.
- FIG. 10A is a process diagram showing a method for manufacturing a calibration sample according to the second embodiment, and is a diagram showing a first layer forming step.
- FIG. 10 is a process diagram showing a method for manufacturing a calibration sample according
- FIG. 4 is a schematic diagram of a microscope image when the focus position is in the vicinity of the interface between the first layer and the second layer and the target object is not in focus.
- FIG. 4 is a diagram showing a contrast graph of images obtained by scanning the objective lens in the optical axis direction a plurality of times, and a focus signal intensity graph used for feedback control of autofocus, with respect to the optical axis direction; be.
- FIG. 14 is a schematic diagram showing a method for manufacturing a calibration sample according to the third embodiment, and shows a state in which a target object is arranged in a sample container;
- FIG. 10A is a process diagram showing a method for manufacturing a calibration sample according to the third embodiment, and is a diagram showing a first layer forming step.
- FIG. 10A is a process diagram showing a method for manufacturing a calibration sample according to the third embodiment, and is a diagram showing a second layer forming step.
- FIG. 1A is a perspective view showing the calibration sample 10 of the first embodiment.
- the calibration sample 10 is used for calibrating the autofocus target position in the optical microscope 20 (FIG. 3A).
- the calibration sample 10 is placed over the first layer 130 along the direction of the optical axis 208 (FIG. 1B) and placed on the side of the bottom surface 120 of the sample holder 110 (eg, hole). and a second layer 140 to be applied.
- FIG. 1B is a cross-sectional view showing the calibration sample 10 of the first embodiment.
- the first layer 130 is obtained by arranging a target object 131 having contrast with respect to the first resin 132 in a first resin 132 that is light transmissive.
- the target object 131 is an object to be focused during autofocusing of the optical microscope 20 (FIG. 3A), details of which will be described later.
- the sample container 100 containing the biological sample for example, culture solution
- FIG. As a result, it is possible to simulate a state in which the biological sample contains the solvent, so that the image quality can be made equivalent to that obtained when the biological sample is observed with an optical microscope.
- the first layer 130 has the same thickness as the size of the target object 131 (the length in the direction of the optical axis 208). Strictly speaking, the first layer 130 has a three-dimensional structure because the target object 131 is tangible. ”.
- the second layer 140 is composed of a light-transmitting second resin 141 .
- the second layer 140 By providing the second layer 140, if the second layer 140 were not provided, the light incident on the first layer 130 from the bottom surface 120 would be interface) can be suppressed. As a result, especially when the first layer 130 is thin, the interference between the reflected light from the upper surface of the first layer 130 and the reflected light from the interface between the first layer 130 and the bottom surface 120 can be suppressed, and autofocus can be performed. It can be done easily.
- the second layer 140 does not include a target object (not shown) that has contrast with the second resin 141 .
- the calibration sample 10 includes a light-transmissive resin sample container 100 containing a first layer 130 and a second layer 140 .
- the sample to be observed (not shown; for example, a biological sample such as a culture solution) can be calibrated using the same sample container as the sample container 100 used for observation.
- the sample container 100 has a plurality of sample holders 110 capable of accommodating observation target samples to be observed with the optical microscope 20 (FIG. 3A). Therefore, the first layer 130 and the second layer 140 can be accommodated in some of the sample holders 110, and the sample to be observed can be accommodated in the remaining sample holders 110.
- the sample container 100 is, for example, a 96-well plate made of colorless and transparent light-transmitting resin.
- the first resin 132 is preferably a thermosetting resin.
- a thermosetting resin By using a thermosetting resin, the first layer 130 can be easily cured when the calibration sample 10 is manufactured. Moreover, when manufacturing the first layer 130 using a solvent during manufacturing, the solvent can be volatilized by heating.
- the first resin 132 has a refractive index of, for example, 0.9 ⁇ n3 or more and 1.1 , where n3 is the refractive index of the solvent contained in the observation target sample (not shown) observed by the optical microscope 20 (FIG. 3A).
- the resin preferably has a refractive index of xn3 or less , preferably 0.95 x n3 or more and 1.05 x n3 or less.
- the first resin 132 when the biological sample contains water as a solvent, the first resin 132 has a refractive index of 1.33, which is the refractive index of water. It preferably has the following refractive indices. Specifically, for example, fluorine resin (refractive index 1.29 to 1.35) can be used. However, the first resin 132 does not necessarily have such a refractive index and need not be a thermosetting resin.
- the target object 131 has contrast with the first resin 132 as described above, for example, optical contrast. Although the details will be described later, the autofocus target position is calibrated by focusing the optical microscope 20 (FIG. 3A) on the target object 131 via the bottom surface 120 .
- the target object 131 has, for example, a brightness shade different from that of the first resin 132 in an image or video captured by the optical microscope 20 .
- the target object 131 can be recognized separately from the first resin 132 on the image or video.
- at least one of the refractive index or color of the target object 131 is different from the refractive index or color of the first resin 132 . Accordingly, the behavior of light is changed between the target object 131 and the first resin 132, and the target object 131 can be distinguished from the first resin 132 and recognized.
- the target object 131 has, for example, a shape (including size) corresponding to an observation target sample (not shown) observed by the optical microscope 20 .
- the calibration sample 10 simulating the sample to be observed can be produced.
- the sample to be observed is, for example, a culture solution containing bacteria or cells
- the target object 131 can be particles such as spheres and ellipsoids.
- the size for example, cells can be particles having a diameter of about 10 ⁇ m, and bacteria can be particles having a diameter of about 1 ⁇ m.
- the target object 131 is, for example, particles dispersed in the first resin 132 .
- the target objects 131 can be prevented from adhering to each other, and unintended enlargement of the target object 131 can be prevented. It is preferable that the target object 131 be uniformly dispersed over the entire first resin 132 .
- the second resin 141 is light transmissive, such as a UV curable resin.
- a UV curable resin By using the UV curable resin, the influence of heat on the first resin 132 can be suppressed.
- the refractive index of the second layer 140 is preferably close to the refractive index of the first layer 130 from the viewpoint of suppressing reflection of light at the interface between the first layers 130 and the second layers 140 .
- the refractive index of the resin forming at least the bottom surface 120 of the sample container 100 is n 0
- the refractive index of the first resin 132 is n 1
- the refractive index of the second resin 141 is n 0 .
- the ratio is n2, it is preferable to satisfy the following formula (1).
- the light reflectance at the interface between the first layer 130 and the second layer 140 is reduced to 1/100 or less of the light reflectance at the interface between the first layer 130 and the bottom surface 120. can. This can suppress reflection of light incident on the first layer 130 from the bottom surface 120 side at the interface between the first layer 130 and the second layer 140 . Therefore, the reflected light from the bottom surface 120 can be easily identified, and autofocus can be easily performed.
- the thickness of the second layer 140 in the direction of the optical axis 208 is, for example, a length that can attenuate the light that enters the second layer 140 from the first layer 130 and is reflected at the interface between the second layer 140 and air. is preferred. By doing so, the reflected light at the interface between the second layer 140 and the air can be ignored, and autofocus can be easily performed.
- a thickness is, for example, 100 ⁇ m or more
- the upper limit is the thickness from which the upper end of the second layer 140 reaches the upper end (opening) of the sample holding portion 110 .
- FIG. 2A is a process diagram showing the method of manufacturing the calibration sample 10 of the first embodiment, and shows a state in which the first resin 132 with the target object 131 dispersed therein is placed in the sample holder 110.
- FIG. A first resin 132 mixed with a target object 131 is applied to the surface of the bottom surface 120 of the sample holder 110 .
- the applied first resin 132 is a fluid before hardening. Therefore, it is preferable to apply the first resin 132 before curing with the target object 131 sufficiently mixed and dispersed in advance.
- the fluid before curing of the first layer 130 can be diluted with a volatile solvent.
- a volatile solvent By diluting with a volatile solvent, the fluid before hardening of the first layer 130 is applied, and then the solvent is volatilized by heating or applying negative pressure to obtain the fluid before hardening of the first layer 130. can be made into a film thickness of about several ⁇ m.
- FIG. 2B is a process diagram showing the manufacturing method of the calibration sample 10 of the first embodiment, and shows the first layer forming process.
- the first layer 130 in which the target object 131 having a contrast with respect to the first resin 132 is arranged in the first resin 132 of the light transmission is formed by using a light-transmitting resin. This is the step of forming on the bottom surface 120 inside the sample container 100 .
- the first resin 132 is, for example, a thermosetting resin
- the first layer 130 can be formed by curing by applying heat.
- a solvent is used, it may be volatilized in advance and then cured, or in the case of curing by heating, it may be volatilized while curing.
- FIG. 2C is a process diagram showing the manufacturing method of the calibration sample 10 of the first embodiment, and shows the second layer forming process.
- a second layer 140 made of a light-transmitting second resin 141 is formed, and the first layer 130 is formed along the direction of the optical axis 208 (FIG. 3A) of the optical microscope 20 (FIG. 3A). It is a process of arranging so that it may cover.
- the second layer 140 can be formed, for example, by applying a fluid of UV curable resin before it is cured on the upper surface of the first layer 130 and curing it by irradiating it with ultraviolet rays.
- FIG. 3A is a diagram illustrating a method of calibrating the autofocus target position using the calibration sample 10 of the first embodiment.
- the calibration method shown in FIG. 3A is performed in optical microscope 20 .
- the optical microscope 20 includes an objective lens 201, an actuator 202 that drives the objective lens 201 along the direction of an optical axis 208, an imaging lens (not shown) that acquires a microscope image from light condensed by the objective lens 201, and A camera 205 and an autofocus unit 206 are provided.
- the autofocus unit 206 (first autofocus mechanism) performs optical autofocus based on reflected light caused by light irradiation.
- the autofocus unit 206 includes a light source such as a laser that irradiates the bottom surface 120 and a detection unit that receives reflected light from the bottom surface 120, both of which are not shown. Further, the autofocus unit 206 includes a register (not shown) for recording target focus signal strength for feedback control of autofocus.
- the signal obtained by converting the reflected light intensity into an electric signal by the detector is called the focus signal intensity
- the target value fed back from the signal intensity is called the target focus signal intensity.
- FIG. 3B is a schematic diagram of a microscope image in which the focus position 207 (FIG. 3A) is shifted from the position of the target object 131 (FIG. 3A) toward the bottom surface 120 (FIG. 3A).
- FIG. 3C is a schematic diagram of a state in which the focus position 207 and the position of the target object 131 match and a microscope image with the maximum contrast is obtained.
- FIG. FIG. 2 is a schematic diagram of a microscopic image that is displaced. The images shown in FIGS. 3B to 3D are obtained by scanning the objective lens 201 in the direction of the optical axis 208 and acquiring microscope images of the calibration sample 10 multiple times.
- the in-focus position and the autofocus target position are relative positions from the tip of the objective lens 201 .
- the focus position is determined by the positional relationship between the objective lens 201 or imaging lens (not shown) and the camera sensor (not shown). A clear image can be obtained.
- the contour of the image of the target object 131 in the microscopic image at the focus position 207 in FIG. 3C is clear. Since the target object 131 is out of focus position 207 in the microscopic images of FIGS. 3B and 3D, the image of the target object 131 in the microscopic image is defocused and has an unclear outline. Contrast is generally used as an index indicating the degree of clarity of this contour.
- the optical microscope 20 is provided with a control device 30 (second autofocus mechanism) that acquires a microscope image (image or video) and performs image identification autofocus based on the contrast in the acquired microscope image.
- the control device 30 includes, for example, a CPU (Central Processing Unit), a RAM (Random Access Memory), a ROM (Read Only Memory), etc., although none of them are shown.
- the control device 30 is embodied by developing a predetermined control program stored in the ROM into the RAM and executing it by the CPU.
- FIG. 3E is a graph 310 of the contrast of multiple microscope images acquired by scanning the objective lens 201 (FIG. 3A) in the direction of the optical axis 208, and a graph 320 of the focus signal strength used for autofocus feedback control. are shown with respect to the direction of the optical axis 208.
- FIG. The horizontal axis indicates the position in the direction of the optical axis 208 (FIG. 3A), the left vertical axis indicates the contrast, and the right vertical axis indicates the focus signal intensity.
- the intensity of the focus signal may be the intensity of reflected light generated by irradiating the bottom surface 120 with light.
- the focus signal strength 322 usually matches the position 311 where the contrast is maximum. Therefore, the focus target is set at the position of the focus signal strength 322 .
- autofocus may not operate normally due to, for example, variations in the manufacturing of the optical components that make up the optical microscope 20, variations in assembly thereof, variations in the sample container 100, and the like. Therefore, the autofocus target position is calibrated and a new autofocus target position is set as the target focus signal strength 323 .
- the target focus signal strength 323 is the focus signal strength shown in the graph 320 at the position 311 where the contrast is maximum.
- Target focus signal strength 323 is recorded in a register (not shown) of controller 30 (FIG. 3A). Although there are two positions where the focus signal strength shown in the graph 320 becomes the target focus signal strength 323, one can be determined by calculating the positional differentiation of the focus signal strength. Alternatively, the target focus signal strength 323 may be set to the maximum focus signal strength 322 . In this case, the lens position of the camera 205 (FIG. 3A) or the autofocus unit 206 (FIG. 3A) is adjusted so that the maximum position 321 of the focus signal and the position 311 where the contrast is maximum match.
- controller 30 receives reflected light signals along the direction of optical axis 208 (FIG. 3A) of optical microscope 20 (FIG. 3A) and captures a plurality of the microscope images. Thereby, the target focus signal intensity 323 of the reflected light in the autofocus section 206 (FIG. 3A, first autofocus mechanism) when the contrast by the control device 30 (second autofocus mechanism) is maximized is determined. Then, the control device 30 sets the position corresponding to the target focus signal strength 323 as the autofocus target position. During observation of an observation target sample (not shown), the objective lens 201 (FIG. 3A) is controlled by the autofocus unit 206 so as to reach the newly set autofocus target position.
- the control device 30 determines the target focus signal strength 323 for each of the plurality of calibration samples 10, and sets the autofocus target position by a statistical method based on the plurality of target focus signal strengths 323. preferably.
- the set target focus signal strength 323 is recorded in the register (not shown).
- the statistical method here is, for example, the average value, mode value, or median value of multiple target focus signal intensities 323 .
- the median value is preferable. As a result, variations can be suppressed to the same extent.
- the sample container 100 preferably comprises at least five sample holders 110 (Fig. 1A) containing first layers 130 (Fig. 1A) and second layers 140 (Fig. 1A). Note that FIG. 1A shows only four sample holders 110 accommodating the first layer 130 and the second layer 140 among the plurality of sample holders 110 provided in the sample container 100 . Then, the control device 30 (FIG. 3A) preferably uses at least five sample holders 110 containing the first layer 130 and the second layer 140 as the sample containers 100 and sets autofocus target positions for each of them. As a result, the number of autofocus target positions to be set can be increased, and variations can be further suppressed.
- the sample holders 110 at the four corners and the sample holders 110 near the center are used in order to efficiently calculate the distribution of variations in the optical characteristics of the individual sample containers 100. It is preferable to obtain at least 5 points including
- the vicinity of the center is, for example, the sample holding portion 110 existing at the center of gravity of the sample container 100 (the intersection of the diagonal lines), or the sample holding portion 110 closest to the center of gravity. Accordingly, the autofocus target position can be set in consideration of variations in optical characteristics of the entire sample container 100 .
- the calibration sample 10 it is possible to simulate an observation target sample that does not have a three-dimensional structure, such as when the observation target sample (not shown; for example, a biological sample such as a cell) is localized on the bottom surface of the sample container 100.
- the observation target sample not shown; for example, a biological sample such as a cell
- the focus target position can be set at, for example, the central position of the variation, and the optimum focus position that can obtain the maximum contrast can be set.
- the calibration sample 10 since calibration is performed without using a sample to be observed, it is possible to provide the calibration sample 10 that does not easily change over time without volatilization of the solvent.
- FIG. 4A is a perspective view showing the calibration sample 11 of the second embodiment.
- FIG. 4B is a cross-sectional view showing the calibration sample 11 of the second embodiment.
- the target objects 131 are unevenly distributed in FIG. 4B for convenience of illustration, it is preferable that they are uniformly dispersed in the first resin 132 in practice.
- the second embodiment relates to a calibration sample 10 in which the first layer 130 has a three-dimensional structure.
- the thickness of the first layer 130 has a thickness greater than the size (eg, grain size) of the target object 131 .
- the thickness of the first layer 130 has a thickness approximately three times the size of the target objects 131 (approximately three target objects 131 in the direction of the optical axis 208).
- the thickness of the first layer 130 is not limited to this example, and the first embodiment or the second embodiment can be selected according to, for example, the amount of solvent in the sample to be observed.
- FIG. 5A is a process diagram showing the method of manufacturing the calibration sample 11 of the second embodiment, and shows the first layer forming process.
- the first layer 130 has a thickness and a three-dimensional structure.
- the thickness of the first layer 130 can be adjusted, for example, by changing the concentration of the first resin 132. For example, the higher the concentration, the greater the thickness. On the other hand, the higher the concentration, the higher the viscosity. Therefore, when the concentration is high, a pre-cured fluid diluted with a solvent may be applied. It is preferable that the solvent is sufficiently volatilized after coating and before curing. Note that the fluid may be applied without using the solvent.
- a thermosetting resin for example, can be used for the first resin 132 as in the first embodiment.
- FIG. 5B is a process diagram showing the method of manufacturing the calibration sample 11 of the second embodiment, and shows the second layer forming process. Also in the second embodiment, the second layer forming step (FIG. 2C in the first embodiment) is performed in the same manner as in the first embodiment.
- the second resin 141 is preferably a UV curable resin as in the second embodiment.
- FIG. 6A is a diagram illustrating a method of calibrating the autofocus target position using the calibration sample 11 of the second embodiment. Also in the second embodiment regarding the calibration sample 11 having a steric structure, calibration is performed in the same manner as in the first embodiment (FIG. 3A) regarding the calibration sample 10 having no steric structure.
- FIG. 6B is a schematic diagram of a microscope image in which the focus position 207 (FIG. 6A) is near the bottom surface 120 (FIG. 4A) of the first layer 130 (FIG. 4A) and is out of focus on the target object 131.
- FIG. 6C is a schematic diagram of a microscope image when the focus position 207 is in the first layer 130.
- the target objects 131 are actually uniformly distributed in the first layer 130, so that about the same number of target objects 131 are focused on the entire area of the first layer 130 in the height direction.
- FIG. 6D is a schematic diagram of a microscope image when the focus position 207 is near the interface between the first layer 130 and the second layer 140 and the target object 131 is out of focus.
- the images shown in FIGS. 6B-6D correspond to the images shown in FIGS. 3B-3D above.
- FIG. 6E is a plot 310 of the contrast of multiple microscopic images acquired by scanning the objective lens 201 (FIG. 6A) in the direction of the optical axis 208 (FIG. 6A) and the focus signal used for autofocus feedback control.
- FIG. 3B shows each of the intensity graphs 320 against the direction of the optical axis 208.
- FIG. FIG. 6E is the same as FIG. 3E except that the shape of graph 310 is different.
- the target objects 131 are uniformly distributed in the first layer 130, and about the same number of target objects 131 are focused on the entire area of the first layer 130 in the height direction. Therefore, the graph 310 has a wide portion in the height direction (horizontal axis direction).
- the autofocus target position can be calibrated in the same manner as in the first embodiment. Further, in the second embodiment, when calibrating the autofocus target position to the interface between the first layer 130 and the second layer 140, the control device 30 (FIG. 6A) adjusts the target focus signal strength so that the contrast starts to decay. The focus signal strength at the position 311 where the target focus signal strength is the target focus signal strength 323 .
- the sample to be observed is, for example, a biological sample, and a sample to be observed having a three-dimensional structure such as cells dispersed in a culture solution can be simulated.
- the volume of the first layer 130 By adjusting the volume of the first layer 130, the dispersion width of the target object 131 can be controlled, and the height of the three-dimensional structure of the observation target sample can be controlled.
- FIG. 7A is a schematic diagram showing the manufacturing method of the calibration sample 12 (FIG. 7C) of the third embodiment, and shows a state in which the target object 431 is arranged in the sample container 100.
- FIG. 7C the third embodiment simulates an observation target (not shown, for example, a biological sample) that has no three-dimensional structure or almost no three-dimensional structure.
- the target object 431 is arranged in advance on the bottom surface 420 of the sample holder 410 of the sample container 100, for example.
- the target object 431 is, for example, a metal pattern arranged on the inner bottom surface 420 (inner bottom surface) of the sample container 100 .
- Metal patterns can be formed, for example, by metal deposition, lithography, or other shaping techniques. By changing the thickness of the metal pattern, the presence or absence of the three-dimensional structure can be changed.
- the target object 431 the above explanation regarding the target object 131 can be similarly applied.
- FIG. 7B is a process diagram showing the manufacturing method of the calibration sample 12 (FIG. 7C) of the third embodiment, and shows the first layer forming process.
- the fluid of the first resin 132 before curing is applied so as to cover the target object 431 .
- the first resin 132 to be used is preferably a thermosetting resin as in the first embodiment. Curing after application forms the first layer 130 in which the target object 431 is arranged in the first resin 132 .
- FIG. 7C is a process diagram showing the manufacturing method of the calibration sample 12 (FIG. 7C) of the third embodiment, and shows the second layer forming process.
- the second layer 140 can be formed, for example, in the same manner as in the first embodiment described above.
- the second resin 141 is preferably a UV curable resin.
- An optical microscope equipped with a first autofocus mechanism based on reflected light generated due to light irradiation and a second autofocus mechanism based on the contrast in an acquired microscope image, and using a calibration sample A calibration device for a focus target position, The calibration sample is Along the optical axis direction of the optical microscope, a first layer arranged on the bottom surface side, in which a target object having a contrast with respect to the first resin is arranged in a light-transmitting first resin; a second layer arranged to cover the first layer and made of a second light-transmitting resin; Equipped with a light-transmitting resin sample container containing In the first autofocus mechanism when the contrast of the second autofocus mechanism is maximized by receiving the signal of the reflected light and capturing a plurality of the microscope images along the optical axis direction of the optical microscope.
- An apparatus for calibrating an autofocus target position wherein a target focus signal intensity of the reflected light is determined, and a position corresponding to the target focus signal intensity is set as the autofocus target position.
- the target focus signal strength is determined for each of the plurality of calibration samples, and the autofocus target position is set by a statistical method based on the plurality of target focus signal strengths.
- autofocus target position calibration device (Appendix 3) The autofocus target position calibrating device according to appendix 2, wherein the statistical method is an average value, mode value, or median value of the plurality of target focus signal intensities.
- the sample container comprises at least five sample holders containing the first layer and the second layer; Any one of Appendices 1 to 3, wherein at least five sample holders containing the first layer and the second layer are used as the sample containers, and the autofocus target positions are set respectively. 3.
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Abstract
Description
(付記1)
光の照射に起因して生じた反射光に基づく第1オートフォーカス機構と、取得した顕微鏡像でのコントラストに基づく第2オートフォーカス機構とを備える光学顕微鏡に備えられ、校正用サンプルを用いたオートフォーカス目標位置の校正装置であって、
前記校正用サンプルは、
前記光学顕微鏡の光軸方向に沿って、
底面側に配置され、光透過性の第1樹脂中に前記第1樹脂に対してコントラストを有する対象物体を配置した第1層と、
前記第1層を覆って配置され、光透過性の第2樹脂により構成される第2層と、
を収容した光透過性の樹脂製のサンプル容器を備え、
前記光学顕微鏡の光軸方向に沿って前記反射光の信号の受信及び複数枚の前記顕微鏡像の撮像により、前記第2オートフォーカス機構によるコントラストが最大となるときの、前記第1オートフォーカス機構における前記反射光のターゲットフォーカス信号強度を決定し、前記ターゲットフォーカス信号強度に対応する位置をオートフォーカス目標位置に設定する
ことを特徴とするオートフォーカス目標位置の校正装置。
(付記2)
複数の前記校正用サンプルのそれぞれについて前記ターゲットフォーカス信号強度を決定し、複数の前記ターゲットフォーカス信号強度に基づく統計学的手法により、前記オートフォーカス目標位置に設定する
ことを特徴とする付記1に記載のオートフォーカス目標位置の校正装置。
(付記3)
前記統計学的手法は、複数の前記ターゲットフォーカス信号強度の平均値、最頻値、又は中央値である
ことを特徴とする付記2に記載のオートフォーカス目標位置の校正装置。
(付記4)
前記サンプル容器は、前記第1層及び前記第2層を収容した少なくとも5つのサンプル保持部を備え、
前記第1層及び前記第2層を収容した少なくとも5つの前記サンプル保持部を前記サンプル容器として用いて、それぞれ前記オートフォーカス目標位置を設定する
ことを特徴とする付記1~3の何れか1項に記載のオートフォーカス目標位置の校正装置。
100 サンプル容器
110,410 サンプル保持部
120,420 底面
130 第1層
131,431 対象物体
132 第1樹脂
140 第2層
141 第2樹脂
20 光学顕微鏡
201 対物レンズ
202 アクチュエータ
205 カメラ
206 オートフォーカス部(第1オートフォーカス機構)
207 合焦位置
208 光軸
30 制御装置(第2オートフォーカス機構、オートフォーカス目標位置の校正装置)
310,320 グラフ
311,313 位置
322 フォーカス信号強度
323 ターゲットフォーカス信号強度
Claims (15)
- 光学顕微鏡におけるオートフォーカス目標位置の校正用サンプルであって、
前記光学顕微鏡の光軸方向に沿って、
底面側に配置され、光透過性の第1樹脂中に前記第1樹脂に対してコントラストを有する対象物体を配置した第1層と、
前記第1層を覆って配置され、光透過性の第2樹脂により構成される第2層と、
を収容した光透過性の樹脂製のサンプル容器を備える
ことを特徴とする校正用サンプル。 - 前記第1樹脂は熱硬化性樹脂である
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 前記第1樹脂は、前記光学顕微鏡により観察される観察対象サンプルに含まれる溶媒の屈折率をn3としたときに、0.9×n3以上1.1×n3以下の屈折率を有する樹脂である
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 前記第2樹脂はUV硬化樹脂である
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 前記対象物体は、前記光学顕微鏡により撮影された画像又は映像において、前記第1樹脂とは異なる輝度の濃淡を有する
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 前記対象物体の屈折率又は色の少なくとも一方は、前記第1樹脂の屈折率又は色とは異なる
ことを特徴とする請求項6に記載の校正用サンプル。 - 前記対象物体は、前記光学顕微鏡により観察される観察対象サンプルに対応する形状を有する
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 前記対象物体は、前記第1樹脂中に分散した粒子である
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 前記対象物体は、前記サンプル容器の内側底面に配置された金属パターンである
ことを特徴とする請求項1又は2に記載の校正用サンプル。 - 光学顕微鏡におけるオートフォーカス目標位置の校正用サンプルの製造方法であって、
光透過性の第1樹脂中に前記第1樹脂に対してコントラストを有する対象物体を配置した第1層を、光透過性の樹脂製のサンプル容器の内側底面上に形成する第1層形成工程と、
光透過性の第2樹脂により構成される第2層を、前記光学顕微鏡の光軸方向に沿って前記第1層を覆うように配置する第2層形成工程と、を含む
ことを特徴とする校正用サンプルの製造方法。 - 光の照射に起因して生じた反射光に基づく第1オートフォーカス機構と、取得した顕微鏡像でのコントラストに基づく第2オートフォーカス機構とを備える光学顕微鏡において、校正用サンプルを用いたオートフォーカス目標位置の校正方法であって、
前記校正用サンプルは、
前記光学顕微鏡の光軸方向に沿って、
底面側に配置され、光透過性の第1樹脂中に前記第1樹脂に対してコントラストを有する対象物体を配置した第1層と、
前記第1層を覆って配置され、光透過性の第2樹脂により構成される第2層と、
を収容した光透過性の樹脂製のサンプル容器を備え、
前記光学顕微鏡の光軸方向に沿って前記反射光の信号の受信及び複数枚の前記顕微鏡像の撮像により、前記第2オートフォーカス機構によるコントラストが最大となるときの、前記第1オートフォーカス機構における前記反射光のターゲットフォーカス信号強度を決定し、前記ターゲットフォーカス信号強度に対応する位置をオートフォーカス目標位置に設定する
ことを特徴とするオートフォーカス目標位置の校正方法。 - 複数の前記校正用サンプルのそれぞれについて前記ターゲットフォーカス信号強度を決定し、複数の前記ターゲットフォーカス信号強度に基づく統計学的手法により、前記オートフォーカス目標位置に設定する
ことを特徴とする請求項12に記載のオートフォーカス目標位置の校正方法。 - 前記統計学的手法は、複数の前記ターゲットフォーカス信号強度の平均値、最頻値、又は中央値である
ことを特徴とする請求項13に記載のオートフォーカス目標位置の校正方法。 - 前記サンプル容器は、前記第1層及び前記第2層を収容した少なくとも5つのサンプル保持部を備え、
前記第1層及び前記第2層を収容した少なくとも5つの前記サンプル保持部を前記サンプル容器として用いて、それぞれ前記オートフォーカス目標位置を設定する
ことを特徴とする請求項12~14の何れか1項に記載のオートフォーカス目標位置の校正方法。
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JP2008215824A (ja) * | 2007-02-28 | 2008-09-18 | Hitachi High-Technologies Corp | 荷電粒子ビーム測長装置、この装置での寸法校正方法、及び校正用標準材 |
JP2013160815A (ja) * | 2012-02-01 | 2013-08-19 | Olympus Corp | 顕微鏡 |
JP2018000102A (ja) * | 2016-07-01 | 2018-01-11 | 富士フイルム株式会社 | 撮影装置および方法並びに撮影制御プログラム |
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JPH1073414A (ja) * | 1996-09-02 | 1998-03-17 | Olympus Optical Co Ltd | 光学調整用標本 |
JP2006275964A (ja) * | 2005-03-30 | 2006-10-12 | Olympus Corp | 走査型蛍光顕微鏡のシェーディング補正方法 |
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