WO2017145487A1 - 顕微鏡および観察方法 - Google Patents
顕微鏡および観察方法 Download PDFInfo
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- WO2017145487A1 WO2017145487A1 PCT/JP2016/086151 JP2016086151W WO2017145487A1 WO 2017145487 A1 WO2017145487 A1 WO 2017145487A1 JP 2016086151 W JP2016086151 W JP 2016086151W WO 2017145487 A1 WO2017145487 A1 WO 2017145487A1
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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
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/241—Devices for focusing
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/241—Devices for focusing
- G02B21/244—Devices for focusing using image analysis techniques
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/241—Devices for focusing
- G02B21/245—Devices for focusing using auxiliary sources, detectors
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/248—Base structure objective (or ocular) turrets
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/24—Base structure
- G02B21/26—Stages; Adjusting means therefor
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/365—Control or image processing arrangements for digital or video microscopes
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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
- G02B7/36—Systems for automatic generation of focusing signals using image sharpness techniques, e.g. image processing techniques for generating autofocus signals
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03B—APPARATUS OR ARRANGEMENTS FOR TAKING PHOTOGRAPHS OR FOR PROJECTING OR VIEWING THEM; APPARATUS OR ARRANGEMENTS EMPLOYING ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ACCESSORIES THEREFOR
- G03B13/00—Viewfinders; Focusing aids for cameras; Means for focusing for cameras; Autofocus systems for cameras
- G03B13/32—Means for focusing
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- H—ELECTRICITY
- H04—ELECTRIC COMMUNICATION TECHNIQUE
- H04N—PICTORIAL COMMUNICATION, e.g. TELEVISION
- H04N23/00—Cameras or camera modules comprising electronic image sensors; Control thereof
- H04N23/60—Control of cameras or camera modules
- H04N23/67—Focus control based on electronic image sensor signals
Definitions
- the detection signal based on the reflected light L1 from the observation target installation surface P1 and the culture liquid cannot be detected separately, and the observation target installation surface P1 may not be focused.
- the depth of focus becomes deep, and it may be difficult to focus on the observation target installation surface P1.
- the present invention provides a microscope and an observation method that can appropriately focus on an observation target installation surface without being affected by the type of culture vessel, the amount of the culture medium, and the magnification of the objective lens.
- the purpose is to do.
- the autofocus control unit searches for a focus position that focuses on the observation target installation surface that is a boundary surface between the bottom of the container and the observation target, based on the focus control information.
- Autofocus control and second autofocus control for searching for a focus position that focuses on the bottom surface of the bottom of the container opposite to the observation target installation surface can be switched.
- the autofocus control unit estimates an in-focus position to be focused on the observation target installation surface by adding an offset to the in-focus position searched by the second autofocus control. Then, the distance changing unit can be controlled based on the estimated focus position.
- the autofocus control unit performs the first autofocus control when the magnification of the objective lens is relatively high, based on the magnification information of the objective lens, When the magnification of the objective lens is relatively low, the second autofocus control can be performed.
- the focusing light has a preset pattern.
- light having a striped pattern can be used as the focusing light.
- the microscope system of the present embodiment includes an illumination light irradiation unit 10, an imaging optical system 30, an imaging unit 40, a focusing light irradiation unit 70, a reflected light detection unit 75, a microscope, and the like.
- a control device 50, a display device 80, and an input device 90 are provided.
- a stage 61 corresponding to a container support unit is provided between the illumination light irradiation unit 10 and the imaging optical system 30, and the culture vessel 60 is placed on the stage 61. And supported.
- a culture solution C and an observation target S that are liquids are accommodated.
- the microscope system includes a stage drive unit 62 that moves the stage 61 in the X direction, the Y direction, and the Z direction.
- the X direction and the Y direction are directions orthogonal to each other on a plane parallel to the observation target installation surface P1, and the Z direction is a direction orthogonal to the X direction and the Y direction.
- the illumination light irradiation unit 10 the imaging optical system 30, the imaging unit 40, the focus light irradiation unit 70, the reflected light detection unit 75, the stage 61, the stage drive unit 62, and the distance change described above.
- the phase contrast microscope main body is constituted by the unit 34, and the microscope control device 50 controls the phase contrast microscope main body.
- the microscope control device 50 controls the phase contrast microscope main body.
- the illumination light irradiation unit 10 irradiates the observation target S accommodated in the culture vessel 60 with illumination light for so-called phase difference measurement.
- illumination for the phase difference measurement is performed.
- a ring-shaped illumination light is irradiated as light.
- the illumination light irradiation unit 10 of the present embodiment has a white light source 11 that emits white light for phase difference measurement and a ring-shaped slit, and white light emitted from the white light source 11 is incident.
- the slit plate 12 that emits the ring-shaped illumination light, the ring-shaped illumination light emitted from the slit plate 12 is incident, and the condenser lens 13 that irradiates the incident ring-shaped illumination light to the observation object S It has.
- the slit plate 12 is provided with a ring-shaped slit that transmits white light to the light-shielding plate that blocks the white light emitted from the white light source 11, and the ring shape is obtained when the white light passes through the slit. Illumination light is formed.
- the ring-shaped illumination light is formed using the slit plate 12 as described above.
- the method for forming the ring-shaped illumination light is not limited to this, for example, spatial light modulation.
- Ring-shaped illumination light may be formed using an element or the like.
- the ring-shaped illumination light is used as the illumination light for phase difference measurement.
- illumination light having a structure other than the ring shape may be used, and the shape is conjugate with the phase plate 32 described later.
- Other shapes such as a triangular shape or a quadrangular shape may be used as long as they are.
- a cell group or the like is arranged as the observation target S.
- the boundary surface between the bottom of the culture vessel 60 and the observation target S is referred to as an observation target installation surface P1.
- the surface opposite to the observation target installation surface P1 at the bottom of the culture vessel 60 is referred to as a bottom surface P2.
- a petri dish and a well plate in which a plurality of wells are arranged can be used. In the case of a well plate, the observation object S and the culture medium C are accommodated in each well.
- the cell group cultured in the culture medium is the observation target S.
- the observation target S is not limited to such a culture target, but water, formalin, ethanol, methanol, and the like.
- the cells fixed in the liquid may be the observation object S.
- the imaging optical system 30 includes an objective lens 31, a phase plate 32, an imaging lens 33, and a distance changing unit 34.
- the phase plate 32 is obtained by forming a phase ring on a transparent plate that is transparent with respect to the wavelength of the ring-shaped illumination light. Note that the size of the slit of the slit plate 12 described above is in a conjugate relationship with this phase ring.
- the phase ring is a ring in which a phase film that shifts the phase of incident light by a quarter wavelength and a neutral density filter that attenuates incident light are formed.
- the phase ring When the direct light incident on the phase plate 32 passes through the phase ring, the phase is shifted by 1 ⁇ 4 wavelength, and the brightness is weakened.
- most of the diffracted light diffracted by the observation object S passes through the transparent plate portion of the phase plate 32, and its phase and brightness do not change.
- the objective lens 31 is moved in the Z direction by the distance changing unit 34.
- autofocus control is performed by moving the objective lens 31 in the Z direction by the distance changing unit 34, and the contrast of the image captured by the imaging unit 40 is adjusted.
- pattern light is irradiated to the culture vessel 60 by the focus light irradiation unit 70, the reflected light is detected by the reflected light detection unit 75, and autofocus control is performed based on the detection signal. .
- the autofocus control based on the detection signal of the reflected pattern light will be described in detail later.
- the imaging lens 33 receives direct light and diffracted light that have passed through the phase plate 32 and forms an image of these lights on the imaging unit 40.
- the distance changing unit 34 includes a mechanism for moving the objective lens 31 in the Z direction as described above, and corresponds to the distance changing unit of the present invention.
- the focus light irradiation unit 70 is used for autofocus control as described above, and irradiates the culture vessel 60 with pattern light having a preset pattern as focus light. Specifically, the focus light irradiation unit 70 of the present embodiment emits pattern light having a striped pattern, and a near-infrared light source 71 that emits near-infrared light, and a near-infrared light source.
- the pattern light which has the striped light-dark pattern emitted from the grid 72 comprised from the linear part which permeate
- near-infrared light is used as the pattern light.
- the wavelength of the pattern light is not limited to this, and other wavelengths may be used as long as the wavelength is different from the illumination light for phase difference measurement. Good.
- the pattern light having a striped bright and dark pattern is formed by using the grid 72.
- the method of forming the pattern light is not limited to this, for example, spatial light modulation.
- a striped bright and dark pattern may be formed using an element or the like.
- the light and dark pattern included in the pattern light is not limited to the stripe shape, and a lattice pattern in which light and dark patterns are two-dimensionally arranged periodically may be used.
- light in which bright and dark patterns are arranged concentrically or light in which dot patterns are arranged two-dimensionally may be used as pattern light.
- the pattern included in the pattern light may not be a black and white pattern, but may be a color pattern composed of different colors.
- the reflected light detection unit 75 detects reflected light by irradiating the culture vessel 60 with pattern light.
- the reflected light detection unit 75 of this embodiment includes a half mirror 76, an optical path difference prism 77, and a line sensor 78.
- the half mirror 76 transmits the pattern light emitted from the grid 72, and reflects the reflected light from the irradiation of the pattern light onto the culture vessel 60 in the direction of the optical path difference prism 77.
- the optical path difference prism 77 divides the reflected light of the incident pattern light into two optical paths and forms images at two different locations of the line sensor 78.
- the line sensor 78 outputs the first detection signal and the second detection signal imaged at two locations to the autofocus control unit 51 of the microscope control device 50.
- the reflected light of the two optical paths divided by the optical path difference prism 77 is detected by one line sensor 78.
- two sensors that individually detect the reflected light of each optical path are provided. You may make it provide.
- the microscope control device 50 is composed of a computer having a CPU (Central Processing Unit), a semiconductor memory, a hard disk, and the like.
- CPU Central Processing Unit
- the microscope control device 50 controls the entire operation of the phase-contrast microscope main body. Specifically, as shown in FIG. 1, an autofocus control unit 51 that controls the distance changing unit 34 and a stage drive unit 62. A stage control unit 52 that controls the focus control information acquisition unit 53.
- the autofocus control unit 51 performs autofocus control by moving the objective lens 31 in the Z direction based on the first detection signal and the second detection signal detected by the line sensor 78 of the reflected light detection unit 75. Is what you do. Specifically, the autofocus control unit 51 of the present embodiment moves the objective lens 31 to a position where the contrast (waveform pattern) of the first detection signal and the contrast (waveform pattern) of the second detection signal approximate. Thus, the in-focus position is determined.
- the first and second detection signals are detected using the line sensor 78.
- the present invention is not limited to this, and a CMOS image sensor or a CCD image sensor may be used.
- the stage control unit 52 drives and controls the stage drive unit 62, thereby moving the stage 61 in the X direction, the Y direction, and the Z direction.
- moving the stage 61 in the X direction and the Y direction for example, one well is scanned with illumination light for phase difference measurement, and each of a plurality of imaging regions (fields of view) divided in one well is scanned. A phase difference image is captured.
- the focus control information acquisition unit 53 acquires at least one focus control information among the information on the culture vessel 60, the information on the amount of the culture medium C, and the information on the magnification of the objective lens 31.
- the information on the culture vessel 60 may be any information as long as it is information related to the thickness of the bottom of the culture vessel 60, for example, information indicating the type of the culture vessel 60 such as the name, manufacturer, or model number of the culture vessel 60. Alternatively, numerical information directly indicating the thickness of the bottom of the culture vessel 60 may be used.
- Information on the culture vessel 60 may be set and input by the user using the input device 90, or a barcode or an IC (Integrated Circuit) chip on which information on the culture vessel 60 is recorded with respect to the culture vessel 60.
- a recording medium may be provided and read out from the recording medium.
- the input device 90 corresponds to the setting input receiving unit of the present invention.
- Information on the amount of the culture medium C may be set and input by the user using the input device 90, or a weight sensor (not shown) is provided on the stage 61 and the like, and the weight is measured by the gravity sensor.
- the amount of the culture medium C may be calculated.
- the information on the amount of the culture solution C is acquired as information that indirectly represents the depth of the culture solution C.
- the magnification information of the objective lens 31 may be set and input by the user using the input device 90, or a barcode or IC (Integrated) in which the magnification of the objective lens 31 is recorded with respect to the objective lens 31.
- a recording medium such as a (Circuit) chip may be provided and read out from the recording medium.
- the focus control information acquired by the focus control information acquisition unit 53 is acquired by the autofocus control unit 51, and the autofocus control unit 51 changes the autofocus control method based on the input focus control information.
- the autofocus control unit 51 of the present embodiment switches between the first autofocus control and the second autofocus control based on the input focus control information.
- the first autofocus control and the second autofocus control will be described.
- the autofocus control unit 51 basically performs autofocus control based on a detection signal of reflected light by irradiation of pattern light. As described above, as a reflection surface on which pattern light is reflected, FIG. There are an observation target installation surface P1, a bottom surface P2 of the culture vessel 60 and a liquid surface P3 of the culture medium C as shown in FIG.
- the autofocus control unit 51 sequentially acquires the first detection signal and the second detection signal of the reflected light of the pattern light described above while moving the objective lens 31 in the Z direction, and the contrast (waveform) of these detection signals.
- the focus position is searched by sequentially determining whether or not (pattern) is approximate.
- the focal position of the objective lens 31 is moved within the range of the arrow R shown in FIG. 3, the focal position is on each surface of the observation target installation surface P1, the bottom surface P2 of the culture vessel 60, and the liquid surface P3 of the culture medium C.
- the focal position is on each surface of the observation target installation surface P1, the bottom surface P2 of the culture vessel 60, and the liquid surface P3 of the culture medium C.
- the autofocus control unit 51 can determine that each surface of the observation target installation surface P1, the bottom surface P2 of the culture vessel 60, and the liquid level P3 of the culture medium C is in focus, that is, the observation target The installation surface P1 can be focused.
- the focal position of the objective lens 31 is moved within the range of the arrow R shown in FIG. 3, it is based on the reflected light L1 from the observation target installation surface P1. Since the detection time point of the detection signal and the detection time point of the detection signal based on the reflected light L2 from the bottom surface P2 of the culture vessel 60 are very close to each other, these detection signals cannot be separated and detected.
- the installation surface P1 cannot be focused.
- the focal position of the objective lens 31 is moved within the range of the arrow R shown in FIG. Since the detection time point of the detection signal based on the reflected light L1 from the observation target installation surface P1 and the detection time point of the detection signal based on the reflected light L3 from the liquid surface P3 of the culture medium C are very close to each other, The detection signals cannot be separated and cannot be focused on the observation target installation surface P1.
- the autofocus control unit 51 of the present embodiment sets the in-focus position to focus on the observation target installation surface P1 when the thickness d of the bottom of the culture vessel 60 is equal to or greater than a preset thickness threshold value.
- the first autofocus control to be searched is performed, and when the thickness d of the bottom of the culture vessel 60 is less than the thickness threshold value, the second focus control different from the first focus control is performed.
- the autofocus control unit 51 performs the first autofocus control described above when the amount of the culture solution C is equal to or greater than a preset liquid threshold value, and the amount of the culture solution C is less than the liquid threshold value. In this case, second auto focus control different from the first focus control is performed.
- the autofocus control unit 51 searches for a focus position that focuses on the bottom surface P2 of the culture vessel 60 and adds an offset to the searched focus position. Thus, the in-focus position for focusing on the observation target installation surface P1 is estimated.
- the detection signal based on the reflected light L1 from the observation target installation surface P1 and the detection signal based on the reflected light L2 from the bottom surface P2 of the culture vessel 60 are separated.
- the bottom surface P2 of the culture vessel 60 does not depend on the state of the observation target S or the like, the reflected light from the bottom surface P2 rather than the detection signal based on the reflected light L1 from the observation target installation surface P1.
- the detection signal based on L2 can be detected with higher accuracy.
- a focus position for focusing on the bottom surface P2 of the culture vessel 60 is searched, and the searched focus position corresponds to the thickness d of the bottom of the culture vessel 60.
- the offset is desirably acquired from the information on the culture vessel 60 acquired as the focus control information.
- the present invention is not limited to this, and a preset value may be used. Settings may be input using the device 90.
- the objective lens 31 is moved so that the focal position of the objective lens 31 comes to the in-focus position estimated as described above.
- the autofocus control unit 51 switches between the above-described first autofocus control and second autofocus control based on the magnification of the objective lens 31. Specifically, when the magnification of the objective lens 31 is a relatively high magnification, the first autofocus control is performed, and when the magnification of the objective lens 31 is a relatively low magnification, the second autofocus control is performed. Autofocus control is performed. Specifically, for example, when the 20 ⁇ objective lens 31 is used, the first autofocus control is performed, and when the 4 ⁇ objective lens 31 is used, the second autofocus control is performed.
- the magnification at which the magnification of the objective lens 31 is relatively high is a magnification higher than 4 times
- the relatively low magnification is a magnification of 4 times or less.
- the reason why the first autofocus control and the second autofocus control are switched depending on the magnification of the objective lens 31 is that the depth of focus differs depending on the magnification of the objective lens 31. That is, when the magnification of the objective lens 31 is relatively high, since the focal depth of the objective lens 31 is shallow, the in-focus position of the observation target installation surface P1 can be searched with high accuracy. This is because, when the magnification of the lens 31 is relatively low, the focal depth of the objective lens 31 is deep, so that the in-focus position of the observation target installation surface P1 cannot be searched with high accuracy.
- second autofocus control for searching for a focus position for focusing on the bottom surface P2 of the culture vessel 60 is performed.
- an input device 90 and a display device 80 are connected to the microscope control device 50.
- the input device 90 includes an input device such as a keyboard and a mouse, and accepts a setting input by a user.
- the input device 90 in the present embodiment accepts setting inputs such as the information on the culture vessel 60 described above, information on the amount of the culture medium C, and information on the magnification of the objective lens 31.
- the display device 80 includes a display device such as a liquid crystal display, and displays a phase difference image captured by the imaging unit 40. In addition, you may make it also serve as the input device 90 by comprising the display apparatus 80 with a touch panel.
- the culture container 60 containing the observation object S and the culture medium C is placed on the stage 61 (S10). Then, focus control information including information on the culture vessel 60, information on the amount of the culture medium C, and information on the magnification of the objective lens 31 is acquired (S12).
- the pattern light is irradiated from the stage 61 side through the objective lens 31 by the focusing light irradiation unit 70 (S14), and the reflected light due to the irradiation of the pattern light is detected by the reflected light detection unit 75 (S16). . Then, the detection signal detected by the reflected light detection unit 75 is input to the autofocus control unit 51.
- the autofocus control unit 51 performs autofocus control based on the input detection signal. At this time, the first autofocus control and the second autofocus control are performed based on the input focus control information. Switch.
- the thickness of the bottom of the culture vessel 60 is equal to or greater than a preset thickness threshold value (S18, YES), and the amount of the culture solution C is equal to or greater than a preset liquid threshold value. If (S20, YES) and the magnification of the objective lens 31 is set to a relatively high magnification (S22, YES), the first autofocus control is performed (S24).
- the phase difference image of the observation target S imaged by the imaging unit 40 is output to the microscope control device 50, and the microscope control device 50 causes the display device 80 to display the input phase difference image.
- focus control information including at least one of information on the culture vessel 60, information on the amount of the culture medium C, and information on the magnification of the objective lens 31 is acquired, and the acquired focus Since the autofocus control method is changed based on the control information, the autofocus control can be performed in consideration of the type of the culture vessel 60, the amount of the culture medium C, and the magnification of the objective lens 31. It is possible to appropriately focus on the observation target installation surface P1.
- the user acquired the information of the thickness of the culture container 60 by setting and inputting the information of the culture container 60, for example, as shown in FIG.
- a thickness measuring unit 100 such as a laser displacement meter for measuring the thickness is provided, and the focus control information acquiring unit 53 acquires the thickness information measured by the thickness measuring unit 100 as information on the culture vessel 60. Also good.
- the above-described autofocus control is performed for each imaging region when a phase difference image for each of the plurality of imaging regions divided in one well of the culture vessel 60 is captured. Can also be done. That is, in the present embodiment, when the stage 61 moves in the X direction and the Y direction, for example, one well as the culture vessel 60 is scanned with illumination light for phase difference measurement and divided in one well. The phase difference image for each of the plurality of imaging regions is captured, but the thickness information of the bottom of the culture vessel 60 for each imaging region measured by the thickness measurement unit 100 is acquired in advance as information on the culture vessel 60. The above-described autofocus control is performed for each imaging region. Thereby, even if the thickness of the bottom of the culture vessel 60 varies within one well, autofocus control can be performed according to the thickness, and the image quality of the phase difference image can be improved.
- information on the amount of the culture medium C information on the culture period of the observation target S may be acquired. Since the amount of the culture medium C evaporates and decreases as the culture period becomes longer, a table in which the culture period and the amount of the culture medium C are associated with each other is set in advance, and the culture period or timer set and input by the user, etc. May be acquired as information on the amount of the culture medium C, and the amount of the culture medium may be acquired with reference to the table.
- information on the amount of the culture medium C information on the depth of the culture medium C may be acquired using the above-described thickness measuring unit 100.
- the phase difference image formed by the imaging optical system 30 is picked up by the image pickup unit 40.
- the image pickup optical system 30 forms an image without providing the image pickup unit 40.
- An observation optical system or the like may be provided so that the user can directly observe the phase difference image of the observed object.
- the present invention is applied to a phase contrast microscope.
- the present invention is not limited to the phase contrast microscope, and is applied to other microscopes such as a differential interference microscope and a bright field microscope. Also good.
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Abstract
Description
11 白色光源
12 スリット板
13 コンデンサレンズ
30 結像光学系
31 対物レンズ
32 位相板
33 結像レンズ
34 距離変更部
40 撮像部
50 顕微鏡制御装置
51 オートフォーカス制御部
52 ステージ制御部
53 フォーカス制御情報取得部
60 培養容器
61 ステージ
62 ステージ駆動部
70 フォーカス用光照射部
71 近赤外光源
72 グリッド
73 照射レンズ
74 ダイクロイックミラー
75 反射光検出部
76 ハーフミラー
77 光路差プリズム
78 ラインセンサ
80 表示装置
90 入力装置
90 入力装置
100 厚さ計測部
C 培養液
L1 反射光
L2 反射光
L3 反射光
P1 観察対象設置面
P2 底面
P3 液面
S 観察対象
Claims (12)
- 液体および観察対象が収容された容器に対して照明光を照射する照明光照射部と、
前記容器が載置され、該容器を支持する容器支持部と、
前記容器および前記容器支持部を通過した前記照明光が入射される対物レンズと、
前記照明光と波長が異なるフォーカス用の光を、前記対物レンズを介して前記容器支持部側から照射するフォーカス用光照射部と、
前記容器支持部側からの前記フォーカス用の光の照射による反射光を、前記対物レンズを介して検出する反射光検出部と、
前記対物レンズと前記容器保持部との距離を変更する距離変更部と、
前記反射光検出部によって検出された反射光に基づいて前記距離変更部を制御することよってオートフォーカス制御を行うオートフォーカス制御部と、
前記容器の情報、前記液体の量の情報および前記対物レンズの倍率の情報のうちの少なくとも1つを含むフォーカス制御情報を取得するフォーカス制御情報取得部とを備え、
前記オートフォーカス制御部が、前記フォーカス制御情報に基づいて、前記オートフォーカス制御の方法を変更することを特徴とする顕微鏡。 - 前記オートフォーカス制御部が、前記フォーカス制御情報に基づいて、前記容器の底部と前記観察対象との境界面である観察対象設置面に合焦する合焦位置を探索する第1のオートフォーカス制御と、前記容器の底部の前記観察対象設置面とは反対側の面である底面に合焦する合焦位置を探索する第2のオートフォーカス制御とを切り替える請求項1記載の顕微鏡。
- 前記オートフォーカス制御部が、前記第2のオートフォーカス制御によって探索された合焦位置に対してオフセットを付加することによって前記観察対象設置面に合焦する合焦位置を推定し、該推定した合焦位置に基づいて前記距離変更部を制御する請求項2記載の顕微鏡。
- 前記オートフォーカス制御部が、前記液体の量の情報に基づいて、前記液体の量が予め設定された液体閾値以上である場合には、前記第1のオートフォーカス制御を行い、前記液体の量が前記液体閾値未満である場合には、前記第2のオートフォーカス制御を行う請求項2または3記載の顕微鏡。
- 前記オートフォーカス制御部が、前記容器の情報に基づいて、前記容器の底部の厚さが予め設定された厚さ閾値以上である場合には、前記第1のオートフォーカス制御を行い、前記容器の底部の厚さが前記厚さ閾値未満である場合には、前記第2のオートフォーカス制御を行う請求項2から4いずれか1項記載の顕微鏡。
- 前記オートフォーカス制御部が、前記対物レンズの倍率の情報に基づいて、前記対物レンズの倍率が相対的に高い倍率である場合には、前記第1のオートフォーカス制御を行い、前記対物レンズの倍率が相対的に低い倍率である場合には、前記第2のオートフォーカス制御を行う請求項2から5いずれか1項記載の顕微鏡。
- 前記フォーカス制御情報取得部が、前記液体の量の情報として、前記観察対象の培養期間の情報を取得する請求項1から6いずれか1項記載の顕微鏡。
- 前記容器の情報として前記容器の底部の厚さを計測する厚さ計測部を備えた請求項1から7いずれか1項記載の顕微鏡。
- 前記フォーカス制御情報の設定入力を受け付ける設定入力受付部を備えた請求項1から8いずれか1項記載の顕微鏡。
- 前記フォーカス用の光が、予め設定されたパターンを有する請求項1から9いずれか1項記載の顕微鏡。
- 前記フォーカス用の光が、縞状のパターンを有する請求項10項記載の顕微鏡。
- 容器支持部に載置された、液体および観察対象が収容された容器に対して照明光を照射し、前記容器および前記容器支持部を通過した前記照明光を対物レンズを介して結像させて前記観察対象の像を観察する観察方法において、
前記照明光と波長が異なるフォーカス用の光を、前記対物レンズを介して前記容器支持部側から照射し、
該フォーカス用の光の照射による反射光を検出し、該検出した反射光に基づいて、前記対物レンズと前記容器保持部との距離を変更することによってオートフォーカス制御を行う際、
前記容器の情報、前記液体の量の情報および前記対物レンズの倍率の情報のうちの少なくとも1つを含むフォーカス制御情報を取得し、
該取得したフォーカス制御情報に基づいて、前記オートフォーカス制御の方法を変更することを特徴とする観察方法。
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