JP5212382B2 - Microscope and aberration correction control method - Google Patents

Description

The present invention relates to a microscope and an aberration correction control method , and more particularly, to a microscope and an aberration correction control method suitable for use in automatically performing aberration correction.

  Conventionally, based on the distance between the sample and the objective lens of the microscope, correction for optical aberration caused by the thickness of the cover glass that protects the sample or the thickness of the holding member that holds the sample and has transparency It has been proposed to correct the aberration by determining the amount and changing the rotation angle of the correction ring of the objective lens in accordance with the control signal based on the determined correction amount (see, for example, Patent Document 1).

JP 2005-31507 A

  By the way, when the immersion objective lens is used for observation, the refractive index of a liquid (hereinafter referred to as an immersion medium) filled between the immersion objective lens and the cover glass changes due to a temperature change, and the refractive index The generated aberration changes due to the change. In particular, an immersion medium having a large refractive index used for an immersion objective lens having a large numerical aperture has a large change in refractive index due to a change in temperature, and a large change in generated aberration. However, the invention described in Patent Document 1 does not consider correcting the change in aberration due to the temperature change of the immersion medium.

  The present invention has been made in view of such a situation, and makes it possible to perform aberration correction more appropriately.

The microscope according to the first aspect of the present invention includes a liquid immersion objective lens including an aberration correction lens, and the temperature of the liquid for immersion filled between the liquid immersion objective lens and the cover glass. and temperature measuring means for measuring, and the aberration correcting means for adjusting the position of the aberration correction lens, by controlling the aberration correcting means to adjust the position of the aberration correction lens, through the immersion objective lens after its control An adjustment value of the aberration correction lens that maximizes the contrast value of the sample image observed in this way is obtained, and the cover is based on the adjustment value and the temperature of the immersion liquid measured by the temperature measuring means. Cover glass thickness detection means for determining the glass thickness, and adjusting the aberration correction lens by controlling the aberration correction means based on the thickness of the cover glass determined by the cover glass thickness detection means And a that control means.

An aberration correction control method according to a second aspect of the present invention is an aberration correction control method for controlling aberration correction of a microscope including an immersion objective lens including an aberration correction lens, the immersion objective lens, a cover glass, A temperature measuring step for measuring the temperature of the immersion liquid filled in between, an aberration correcting step for adjusting the position of the aberration correcting lens, and the position of the aberration correcting lens adjusted by the aberration correcting step. After that, an adjustment value of the aberration correction lens that maximizes the contrast value of the sample image observed through the immersion objective lens is obtained, and the adjustment value and the temperature measurement step are used for the immersion measurement. Cover glass thickness detection step for determining the thickness of the cover glass based on the temperature of the liquid, and the thickness of the cover glass determined in the cover glass thickness detection step Based and a control step of adjusting the aberration correction lens.
An aberration correction control method according to a third aspect of the present invention is an aberration correction control method for controlling aberration correction of a microscope including an immersion objective lens provided with an aberration correction lens, the immersion objective lens and a cover glass. A temperature measurement step for measuring the temperature of the liquid for immersion filled between and the adjustment value data of the aberration correction lens for correcting aberration in the combination of the temperature of the liquid and the thickness of the cover glass Based on the aberration correction step of adjusting the position of the aberration correction lens, and in the state where the thickness of the cover glass is unknown, the thickness of the cover glass different from the temperature of the liquid measured by the temperature measurement step The position of the aberration correction lens is adjusted based on the adjustment value data corresponding to the combination of the above, and observation is performed through the immersion objective lens after the control. Seeking a combination of the thickness of the cover glass contrast value of the specimen image is maximized to, and a control step of adjusting the aberration correction lens at a position corresponding to its thickness.

In the first aspect of the present invention, the temperature of the liquid for filled with and immersion between the immersion objective lens and the cover glass is measured, the position of the aberration correction lens is adjusted, the immersion objective An adjustment value of the aberration correction lens that maximizes the contrast value of the specimen image observed through the lens is obtained, and the thickness of the cover glass is determined based on the adjustment value and the temperature of the immersion liquid. The aberration correction lens is adjusted based on the thickness of the cover glass.
In the second aspect of the present invention, the temperature of the immersion liquid filled between the immersion objective lens and the cover glass is measured, the position of the aberration correction lens is adjusted, and the immersion objective is adjusted. An adjustment value of the aberration correction lens that maximizes the contrast value of the specimen image observed through the lens is obtained, and the thickness of the cover glass is determined based on the adjustment value and the temperature of the immersion liquid. The aberration correction lens is adjusted based on the thickness of the cover glass.
In the third aspect of the present invention, the temperature of the liquid for immersion filled between the immersion objective lens and the cover glass is measured, and in the combination of the temperature of the liquid and the thickness of the cover glass Based on the adjustment value data of the aberration correction lens for correcting the aberration, the position of the aberration correction lens is adjusted, and the thickness of the cover glass is unknown. Based on the adjustment value data corresponding to a combination of different thicknesses, the position of the aberration correction lens is adjusted, and the thickness of the cover glass that maximizes the contrast value of the sample image observed through the immersion objective lens The aberration correction lens is adjusted to a position corresponding to the thickness.

According to the first to third aspects of the present invention, aberration correction can be performed more appropriately.

It is a figure which shows one Embodiment of the microscope to which this invention is applied. It is the figure which showed the structure around the microscope stage and objective lens in detail. It is a block diagram which shows the example of a structure of the function implement | achieved by the control part. It is a flowchart for demonstrating a correction amount table production | generation process. It is a figure which shows the example of a correction amount table. It is a flowchart for demonstrating an automatic aberration correction process.

Explanation of symbols

  1 microscope, 2 Petri dish, 2A cover glass, 14 CCD camera, 21 stage, 22A, 22B objective lens, 24 focusing movable part, 25 focusing part, 71 oil for oil immersion, 72 aberration correction lens, 75 correction ring, 76 Correction ring worm gear, 77 motor, 78 limit plate, 79 limit sensor, 80 stepping motor, 81 rack teeth, 82 linear scale, 83 heat plate, 84 temperature sensor, 92 control unit, 93 storage unit, 101 sample, 151 Temperature measurement unit, 152 rotation angle detection unit, 153 correction amount table generation unit, 154 cover glass thickness detection unit, 155 aberration correction unit, 156 focus control unit

  Embodiments to which the present invention is applied will be described below with reference to the drawings.

  FIG. 1 is a diagram showing an embodiment of a microscope to which the present invention is applied. The main body 11 of the inverted microscope 1 is provided with a transmission illumination device 12 and an epi-illumination device 13, and the specimen in the Petri dish 2 on the stage 21 is observed using transmission illumination or epi-illumination. Can do. Further, a CCD (Charge Coupled Devices) camera 14 is attached to the main body 11 of the microscope 1, and it is possible to take an image of a specimen by transmitted illumination or epi-illumination.

  A focusing unit 25 is provided on the lower surface of the stage 21. The focusing unit 25 moves the focusing movable unit 24 up and down to support the revolver 23 on which a plurality of objective lenses (in FIG. 1, only two objective lenses 22A and 22B are shown) are mounted. Supports to move in the direction. Hereinafter, the objective lenses 22A and 22B are simply referred to as the objective lens 22 when it is not necessary to distinguish them.

  In addition, a plurality of filter blocks each including a combination of two types of filters and a dichroic mirror are provided below the focusing movable unit 24 (in FIG. 1, two filter blocks 16A and 16B are illustrated. ) A filter turret 15 is provided. Hereinafter, the filter blocks 16A and 16B are simply referred to as the filter block 16 when it is not necessary to distinguish them.

  When the specimen is observed with the microscope 1 using the transmission illumination, the transmission illumination light emitted from the light source 31 of the transmission illumination device 12 is transmitted through the collector lens 32 and then reflected toward the condenser lens 34 by the reflection mirror 33. Then, the sample in the Petri dish 2 is irradiated through the condenser lens 34. When the optical path switching prism 52 is removed from the optical path, the transmitted illumination light that has passed through the specimen passes through the objective lens 22, the filter block 16, and the second objective lens 52, and is imaged by the CCD camera 14 with the transmitted illumination light. Is filmed. When the optical path switching prism 52 is inserted in the optical path, the transmitted illumination light transmitted through the sample is transmitted through the objective lens 22, the filter block 16, and the second objective lens 51, and the direction of the prism 53 is transmitted by the optical path switching prism 52. Is reflected in the direction of the eyepiece lens 55 by the prism 53 and the reflecting mirror 54, and the user observes the image of the specimen by the transmitted illumination light through the eyepiece lens 55.

  Further, when the specimen is observed using the epi-illumination by the microscope 1, the epi-illumination light emitted from the light source 41 of the epi-illumination device 13 passes through the relay lenses 42 and 43 and is then transmitted to a predetermined wavelength by the filter block 16. Only is transmitted, is reflected in the direction of the objective lens 22, and is irradiated to the specimen in the Petri dish 2 through the objective lens 22. When the optical path switching prism 52 is removed from the optical path, the fluorescence emitted from the specimen by irradiating the epi-illumination light passes through the objective lens 22, and then only a predetermined wavelength is extracted by the filter block 16, and the CCD camera 14 An image of the fluorescence emitted from the specimen is taken. Further, when the optical path switching prism 52 is placed in the optical path, the fluorescence emitted from the specimen by irradiating the epi-illumination light is transmitted through the objective lens 22, and then only a predetermined wavelength is extracted by the filter block 16, and the optical path Reflected in the direction of the prism 53 by the switching prism 52 and reflected in the direction of the eyepiece lens 55 by the prism 53 and the reflecting mirror 54, the user observes an image of fluorescence emitted from the specimen through the eyepiece lens 55. .

  FIG. 2 is a diagram showing in detail the configuration around the stage 21 and the objective lens 22 of the microscope 1 of FIG. FIG. 2 shows an example in which the culture medium 102 is filled in the Petri dish 2 and the specimen 101 which is a cell to be observed is placed on the cover glass 2A constituting the bottom of the Petri dish 2. ing. In the microscope 1, the temperature of the culture solution 102 can be kept almost constant (for example, 37 ° C.) by the heat plate 83 on the stage 21, and the specimen 101 can be observed while alive.

  An oil immersion oil 71 is filled between the oil immersion objective lens 22A and the cover glass 2A in order to increase the numerical aperture of the optical system and realize high resolution and brightness. The objective lens 22A also includes an aberration correction lens 72 that is used to appropriately correct aberrations that change due to an error in the thickness of the cover glass 2A or a change in refractive index that accompanies a temperature change in the oil immersion oil 71. Yes.

  The aberration correction lens 72 is fixed by a lens frame 73 connected to the cam groove of the correction ring 75 via a pin 74. Therefore, by rotating the correction ring 75 about the axis of the pin 74, the aberration correction lens 72 moves in the optical axis direction together with the lens frame 73, and the position of the aberration correction lens 72 in the optical axis direction can be adjusted. A correction ring worm gear 76 is provided on the outer periphery of the correction ring 75, and a motor 77 with a rotary encoder is driven under the control of the control unit 92, and the correction ring worm gear 76 is corrected. By rotating the ring 75, the aberration correction lens 72 can be electrically moved in the optical axis direction. The control unit 92 detects the rotation angle of the correction ring 75 with reference to the reference position defined by the limit sensor 79 and the limit plate 78 based on the pulse output from the motor 77.

  Further, the focusing unit 25 is provided with a stepping motor 80, and the stepping motor 80 is driven under the control of the control unit 92, and the rack teeth 81 attached to the focusing movable unit 24 are moved in the vertical direction. By moving, the focusing movable unit 24 can be moved in the vertical direction. As a result, the revolver 23 and the objective lens 22 attached to the focusing movable unit 24 move in the vertical direction, and the position of the objective lens 22 used for observation (the objective lens 22A in FIG. 2) in the optical axis direction is electrically driven. Can be adjusted. The control unit 92 detects the position of the objective lens 22 in the optical axis direction based on the position of the rack tooth 81 detected by the linear scale 82.

  By the way, as described above, the heat plate 83 repeats heating and cooling in order to keep the temperature of the culture solution 102 substantially constant and keep the specimen 101 in a good state. Along with this, the temperature of the Petri dish 2 including the cover glass 2A varies, and the temperature of the oil immersion oil 71 filled at the tip of the objective lens 22A also varies due to heat conduction. The controller 92 measures the temperature of the oil immersion oil 71 based on a signal from the temperature sensor 84 installed at the tip of the objective lens 22A, and stores it in the storage unit 93 together with the measurement time.

  The input unit 91 includes, for example, various keys and switches, and is used to input various commands and information to the control unit 92. The control unit 92 includes, for example, a processor such as a CPU (Central Processing Unit), and controls aberration correction of the microscope 1 as will be described later with reference to FIGS. 4 to 6. The control unit 92 is connected to the CCD camera 14 and acquires data indicating the contrast value of an image captured by the CCD camera 14. The storage unit 93 includes, for example, various memories, and stores a correction amount table described later with reference to FIG.

  The input unit 91, the control unit 92, and the storage unit 93 can be provided in the microscope 1, or configured by a dedicated external device or computer other than the microscope 1. It is also possible to do.

  FIG. 3 is a block diagram illustrating an example of a functional configuration realized by the control unit 92 of FIG. 2 executing a predetermined program.

  The temperature measuring unit 151 measures the temperature of the oil for oil immersion 71 based on a signal from the temperature sensor 84. The temperature measurement unit 151 notifies the measured temperature of the oil 71 for oil immersion to the correction amount table generation unit 153, the cover glass thickness detection unit 154, and the aberration correction unit 155 as necessary, or together with the measurement time. Or stored in the storage unit 93.

  The rotation angle detector 152 detects the rotation angle of the correction ring 75 with reference to the reference position defined by the limit sensor 79 and the limit plate 78 based on the pulse output from the motor 77, and if necessary, The correction amount table generation unit 153 and the aberration correction unit 155 are notified.

  As will be described later with reference to FIGS. 4 and 5, the correction amount table generation unit 153 is a combination of the temperature of the oil for oil 71 and the thickness of the cover glass 2 </ b> A input through the input unit 91. A correction amount table, which is data indicating an adjustment value (rotation angle of the correction ring 75) of the position of the aberration correction lens 72 for appropriately correcting the aberration, is generated and stored in the storage unit 93.

  As will be described later with reference to FIG. 6, the cover glass thickness detection unit 154 is notified of the temperature of the oil 71 for oil immersion, the rotation angle of the correction ring 75 notified from the aberration correction unit 155, and the focus control unit 156. The thickness of the cover glass 2A is detected based on the contrast value of the image photographed by the CCD camera 14 and a correction amount table described later with reference to FIG. The cover glass thickness detection unit 154 notifies the aberration correction unit 155 of the detected thickness of the cover glass 2A. Further, the cover glass thickness detection unit 154 notifies the aberration correction unit 155 of an assumed value of the thickness of the cover glass 2A that is set when detecting the thickness of the cover glass 2A.

  As will be described later with reference to FIG. 6, the aberration correction unit 155 determines the thickness of the cover glass 2 </ b> A or the assumed value of the thickness of the cover glass 2 </ b> A, the temperature of the oil 71 for oil immersion, and FIG. 5. Based on a correction amount table to be described later, a rotation angle of the correction ring 75 appropriate for correcting the aberration is obtained. The aberration correction unit 155 controls the motor 77 while monitoring the rotation angle of the correction ring 75 detected by the rotation angle detection unit 152, rotates the correction ring 75 by the calculated rotation angle, and the aberration is appropriately corrected. Thus, the position of the aberration correction lens 72 in the optical axis direction is adjusted. When the adjustment of the position of the aberration correction lens 72 is completed, the aberration correction unit 155 notifies the focus control unit 156 that the adjustment of the position of the aberration correction lens 72 is completed.

  The focus control unit 156 detects the position of the rack tooth 81 based on the signal from the linear scale 82, and the contrast value of the image supplied from the CCD camera 14, that is, the sample observed through the objective lens 22. The focus control of the objective lens 22 is performed by controlling the stepping motor 80 so as to maximize the contrast value of the image and adjusting the position of the objective lens 22 in the optical axis direction.

  Next, processing of the microscope 1 will be described with reference to FIGS. 4 to 6.

  First, correction amount table generation processing executed by the microscope 1 will be described with reference to the flowchart of FIG. This process is started, for example, when the user inputs a correction amount table generation instruction via the input unit 91.

  In step S <b> 1, the temperature measurement unit 151 starts measuring the temperature of the oil immersion oil 71 based on the signal from the temperature sensor 84. The temperature measurement unit 151 notifies the correction amount table generation unit 153 of the measured temperature of the oil 71 for immersion.

  In step S2, the correction amount table generating unit 153 acquires the thickness of the cover glass. By the way, the thickness of the cover glass is allowed to have a manufacturing error within a predetermined range with respect to the standard reference value. For example, an error within the range of 0.15 mm to 0.18 mm is allowed with respect to the standard standard value of 0.17 mm. ing. When generating the correction amount table, the user can create a cover glass having a plurality of known thicknesses within a tolerance range (for example, four types of cover glasses having thicknesses of 0.15 mm, 0.16 mm, 0.17 mm, and 0.18 mm). ). Then, the user installs one of the prepared cover glasses on the stage 21 of the microscope 1 and inputs the thickness of the installed cover glass via the input unit 91. The correction amount table generation unit 153 acquires data indicating the thickness of the cover glass input by the user.

  In step S <b> 3, the correction amount table generation unit 153 determines whether storage of the rotation angle of the correction ring 75 has been commanded. Specifically, the user adjusts the temperature of the heat plate 83 so that the temperature of the oil 71 for oil immersion becomes a desired temperature. When the temperature of the oil immersion oil 71 reaches a desired temperature, the user uses the correction ring 75 to adjust the position of the aberration correction lens 72 in the optical axis direction or adjust the position of the focusing movable unit 24. The focus of the objective lens 22 is adjusted by adjusting the position of the objective lens 22 in the optical axis direction. When it is determined that the focus of the objective lens 22 has been adjusted most appropriately, a command for storing the rotation angle of the correction ring 75 at that time is input via the input unit 91. When the correction amount table generation unit 153 obtains the command, the correction amount table generation unit 153 determines that the storage of the rotation angle of the correction ring 75 has been commanded, and the process proceeds to step S4.

  In step S <b> 4, the correction amount table generation unit 153 sets the rotation angle of the correction ring 75 for the combination of the current cover glass thickness and the temperature of the oil for oil 71 in the correction amount table.

FIG. 5 shows an example of the correction amount table. The correction amount table lists rotation amounts θmn of the correction ring 75 for appropriately correcting aberrations in each combination of the temperature of the oil 71 for immersion and the thickness of the cover glass. In the correction amount table of FIG. 5, the temperature of 16 kinds of oil 71 for oil immersion at intervals of 1 ° C. within a range from 23 ° C. to 39 ° C. and 4 at intervals of 0.01 mm within a range from 0.15 m to 0.18 mm. The rotation amount θmn (m = 1 to 16, n = 1 to 4) of the correction ring 75 is set for each combination with the thickness of the type of cover glass. For example, in FIG. 5, when the temperature of the oil immersion oil 71 is 23 ° C. and the thickness of the cover glass is 0.15 mm, the rotation amount is θ ₁₁ , the temperature of the oil immersion oil 71 is 39 ° C., and the cover glass. rotation amount when the thickness of 0.18mm is theta _164.

  The correction amount table generation unit 153 acquires data indicating the current rotation angle of the correction ring 75 from the rotation angle detection unit 152, and uses the acquired rotation angle of the correction ring 75 as the current cover glass thickness and oil immersion oil. It is set in the column of the correction amount table corresponding to the temperature combination of 71.

  On the other hand, if it is determined in step S3 that the command to store the rotation angle of the correction ring 75 has not been issued, the process of step S4 is skipped, and the process proceeds to step S5.

  In step S5, the correction amount table generation unit 153 determines whether the cover glass has been replaced. If it is determined that the cover glass has not been replaced, the process proceeds to step S6.

  In step S <b> 6, the correction amount table generation unit 153 determines whether the end of the process has been commanded. If it is determined that the end of the process has not been commanded, the process returns to step S3, and it is determined in step S5 that the cover glass has been replaced, or in step S6, it is determined that the end of the process has been commanded. Until then, the processes of steps S3 to S6 are repeated.

  For example, when the thickness of the currently installed cover glass is 0.15 mm, the user adjusts the temperature of the heat plate 83 and changes the temperature of the oil immersion oil 71 in units of 1 ° C. from 23 ° C. to 39 ° C. However, as described above, the focus of the objective lens 22 is adjusted at each temperature, and the rotation angle of the correction ring 75 when the focus is most appropriately adjusted is set in the correction amount table. Then, after the setting of the rotation angle of the correction ring 75 at all temperatures is completed, the user installs the cover glass having the next thickness (for example, 0.16 mm) on the stage 21 and installs it through the input unit 91. Enter the thickness of the cover glass. When the correction amount table generation unit 153 acquires data indicating the thickness of the cover glass input by the user, the correction amount table generation unit 153 determines in step S5 that the cover glass has been replaced, and the process returns to step S2, and the above-described steps S2 and thereafter Is performed.

  Thereafter, as described above, the user changes the temperature of all the oil immersion oils 71 in the correction amount table and the thickness of the cover glass while changing the temperature of the oil immersion oil 71 or replacing the cover glass. The rotation angle of the correction ring 75 for the combination is set. Thereafter, the user inputs an instruction to end generation of the correction amount table via the input unit 91. When the correction amount table generation unit 153 obtains the instruction, it determines that the end of the process is instructed in step S6, and the correction amount table generation process ends.

  When there are a plurality of oil immersion objective lenses having an aberration correction lens, the above-described processing is performed on each objective lens, and a correction amount table for each objective lens is generated.

  In addition, it is not always necessary to perform the correction amount table generation process in all microscopes. For example, a correction amount table for each combination of a microscope and an objective lens is generated by a manufacturer or the like, and the correction amount table is generated. You may make it provide to a user.

  Next, automatic aberration correction processing executed by the microscope 1 will be described with reference to the flowchart of FIG. This process is started, for example, when the user inputs a command for automatic aberration correction processing via the input unit 91 by the user.

  In step S21, the cover glass thickness detector 154 sets an assumed value of the thickness of the cover glass 2A. Specifically, the cover glass thickness detection unit 154 selects one unprocessed thickness among the cover glass thicknesses defined in the correction amount table, and sets the unprocessed thickness to an assumed value of the cover glass 2A. The cover glass thickness detection unit 154 notifies the aberration correction unit 155 of the assumed thickness value of the set cover glass 2A.

  In step S <b> 22, the temperature measurement unit 151 measures the temperature of the oil immersion oil 71 based on the signal from the temperature sensor 84. The temperature measuring unit 151 notifies the measured temperature of the oil immersion oil 71 to the cover glass thickness detecting unit 154 and the aberration correcting unit 155.

In step S <b> 23, the aberration correction unit 155 moves the aberration correction lens 72 to an appropriate position with respect to the assumed value of the thickness of the cover glass 2 </ b> A and the temperature of the oil for oil 71.
Specifically, the aberration correction unit 155 determines the rotation angle of the correction ring 75 with respect to the combination of the current estimated value of the thickness of the cover glass 2A and the temperature of the oil immersion oil 71 based on the correction amount table. In addition, when the column corresponding to the combination of the current estimated value of the cover glass 2A and the temperature of the oil 71 for oil immersion does not exist in the correction amount table, the aberration correction unit 155 uses the values in the other columns to calculate a straight line. The rotation angle of the correction ring 75 is calculated by interpolation.

  The aberration correction unit 155 controls the motor 77 while monitoring the rotation angle of the correction ring 75 detected by the rotation angle detection unit 152, and rotates the correction ring 75 by the calculated rotation angle. Thereby, the position of the aberration correction lens 72 in the optical axis direction is adjusted to an appropriate position with respect to the combination of the current assumed value of the thickness of the cover glass 2A and the temperature of the oil 71 for oil immersion. The aberration correction unit 155 notifies the cover glass thickness detection unit 154 of the set rotation angle of the correction ring 75. The aberration correction unit 155 notifies the focus control unit 156 that the adjustment of the position of the aberration correction lens 72 is completed.

  In step S24, the focus control unit 156 performs focus control. Specifically, the focus control unit 156 adjusts the position of the objective lens 22A in the optical axis direction by controlling the stepping motor 80 so that the contrast value of the image supplied from the CCD camera 14 is maximized. After adjusting the position of the objective lens 22A in the optical axis direction, the focus control unit 156 notifies the cover glass thickness detection unit 154 of the maximum contrast value.

  In step S25, the cover glass thickness detection unit 154 stores the acquired temperature of the oil immersion oil 71, the rotation angle of the correction ring 75, and the maximum value of the contrast value of the image in the storage unit 93 in association with each other.

  In step S <b> 26, the cover glass thickness detection unit 154 determines whether or not the assumed values of the thicknesses of all the cover glasses 2 </ b> A have been processed. If it is determined that the assumed values of the thicknesses of all the cover glasses 2A have not been processed yet, the process returns to step S21, and in step S26, it is determined that the assumed values of the thicknesses of all the cover glasses 2A have been processed. Steps S21 to S26 are repeatedly executed until Thereby, the thicknesses of all the cover glasses defined in the correction amount table are sequentially set as the assumed values of the thickness of the cover glass 2A, and as described above, the temperature of the oil immersion oil 71 at each assumed value, The rotation angle of the correction ring 75 and the maximum contrast value of the image are measured and stored.

  On the other hand, if it is determined in step S26 that processing has been performed for the assumed thickness values of all the cover glasses 2A, the processing proceeds to step S27.

  In step S27, the cover glass thickness detector 154 determines the thickness of the cover glass 2A. Specifically, the cover glass thickness detection unit 154 has a maximum contrast value among combinations of the stored temperature of the oil immersion oil 71, the rotation angle of the correction ring 75, and the maximum value of the contrast value of the image. The combination with the maximum value is extracted. The cover glass thickness detection unit 154 obtains the thickness of the cover glass 2A based on the temperature of the oil immersion oil 71 in the extracted combination, the rotation angle of the correction ring 75, and the correction amount table. More specifically, the cover glass thickness detection unit 154 calculates the cover glass thickness corresponding to the temperature of the oil for oil 71 and the rotation angle of the correction ring 75 in the extracted combination based on the correction amount table. Obtained as a thickness of 2A. The cover glass thickness detection unit 154 notifies the aberration correction unit 155 of the obtained thickness of the cover glass 2A.

  In step S <b> 28, the temperature measurement unit 151 measures the temperature of the oil for oil immersion 71 based on the signal from the temperature sensor 84. The temperature measurement unit 151 notifies the aberration correction unit 155 of the measured temperature of the oil immersion oil 71 and stores it in the storage unit 93 together with the measurement time.

  In step S29, the aberration correction unit 155 determines whether it is time to perform aberration correction. If it is determined that it is time to perform aberration correction, the process proceeds to step S30. The timing for performing the aberration correction is, for example, when a predetermined time has elapsed since the previous aberration correction, or when the temperature of the oil immersion oil 71 has changed by a predetermined value or more after the previous aberration correction, Alternatively, a case where the specimen 101 is photographed by the CCD camera 14 can be considered.

  In step S <b> 30, the aberration correction unit 155 adjusts the position of the aberration correction lens 72 according to the temperature of the oil 71 for oil immersion. Specifically, the aberration correction unit 155 performs the rotation angle of the correction ring 75 with respect to the current combination of the temperature of the oil for oil 71 and the thickness of the cover glass 2A based on the correction amount table by the same processing as in step S23. And the correction ring 75 is rotated by the determined rotation angle. Thereby, the position of the aberration correction lens 72 in the optical axis direction is adjusted to an appropriate position with respect to the current combination of the thickness of the cover glass 2A and the temperature of the oil 71 for oil immersion, and the aberration is appropriately corrected. The aberration correction unit 155 notifies the focus control unit 156 that the adjustment of the position of the aberration correction lens 72 is completed.

  In step S31, the focus control unit 156 performs focus control similarly to the process in step S24. Thereafter, the process proceeds to step S32.

  On the other hand, if it is determined in step S29 that it is not time to perform aberration correction, the processes in steps S29 and S30 are skipped, and the process proceeds to step S32.

  In step S32, the control unit 92 determines whether to end the process. If it is determined not to end the process, the process returns to step S28, and the processes in steps S28 to S32 are repeatedly executed until it is determined in step S32 that the process is to be ended.

  On the other hand, in step S 32, for example, in order to replace the objective lens 22, replace the specimen 101, or end the observation, the user instructs the end of the automatic aberration correction process via the input unit 91. Is input, the control unit 92 determines that the process is to be terminated, and the automatic aberration correction process is terminated.

  In this manner, the thickness of the cover glass 2A can be accurately detected, and appropriate aberration correction can be automatically performed according to the thickness of the cover glass 2A and the temperature of the oil for oil immersion 71. In addition, aberration correction can be automatically performed following the change in the temperature of the oil 71 for immersion.

  If the accurate thickness of the cover glass 2A is known in advance, the thickness can be input to the control unit 92, and the processes of steps S1 to S7 can be omitted.

  In the above description, an example in which an oil immersion objective lens is used has been described. However, even when a water immersion objective lens is used, aberration correction can be performed by the same processing.

  The series of processes described above can be executed by hardware or can be executed by software. When a series of processing is executed by software, a program constituting the software executes various functions by installing a computer incorporated in dedicated hardware or various programs. For example, it is installed from a program recording medium in a general-purpose personal computer or the like.

  The program executed by the computer may be a program that is processed in time series in the order described in this specification, or in parallel or at a necessary timing such as when a call is made. It may be a program for processing.

  Furthermore, the embodiments of the present invention are not limited to the above-described embodiments, and various modifications can be made without departing from the gist of the present invention.

Claims (8)

In a microscope having an immersion objective including an aberration correction lens,
Temperature measuring means for measuring the temperature of the liquid for immersion filled between the immersion objective lens and the cover glass;
And aberration correction means for adjusting the position of the aberration correction lens,
The aberration correction unit is controlled to adjust the position of the aberration correction lens, and the control value of the aberration correction lens that maximizes the contrast value of the specimen image observed through the immersion objective lens after the control is adjusted. Finding the cover glass thickness detecting means for obtaining the thickness of the cover glass based on the adjustment value and the temperature of the immersion liquid measured by the temperature measuring means;
A microscope comprising: control means for adjusting the aberration correction lens by controlling the aberration correction means based on the thickness of the cover glass obtained by the cover glass thickness detection means . An adjustment value of the position of the aberration correction lens when the focus of the aberration correction lens is appropriately adjusted in each of a plurality of combinations of the temperature of the liquid and the thickness of the cover glass is generated , and adjustment value data is generated Adjustment value data generation means ;
Storage means for storing the adjustment value, the temperature of the liquid, and the thickness of the cover glass in association with each other as the adjustment value data generated by the adjustment value data generation means.
Microscope according to claim 1, further comprising a. The cover glass thickness detection means adjusts the position of the aberration correction lens based on each adjustment value corresponding to a combination of different thicknesses of the cover glass with respect to the temperature of the liquid measured by the temperature measurement means. The aberration correction means is controlled to obtain the thickness of the cover glass that maximizes the contrast value of the specimen image observed through the immersion objective lens after the control.
The microscope according to claim 1 or 2, characterized in that The temperature measuring means is a temperature sensor installed at the tip of the immersion objective lens at a position where the temperature measurement unit touches the immersion liquid.
The microscope according to claim 1 . The control means, based on the adjustment value, according to a combination of the temperature of the liquid measured by the temperature measurement means at a predetermined timing and the obtained thickness of the cover glass, Let the aberration correction means adjust the position
The microscope according to any one of claims 1 to 3, characterized by: A focus that adjusts the position of the immersion objective lens so that the contrast value of the image of the specimen observed through the immersion objective lens is maximized after the aberration correction lens is adjusted by the aberration correction means. Control means
The microscope according to claim 1, further comprising: In an aberration correction control method for controlling aberration correction of a microscope including an immersion objective lens including an aberration correction lens,
A temperature measurement step for measuring the temperature of the liquid for immersion filled between the immersion objective lens and the cover glass;
An aberration correction step of adjusting the position of the aberration correction lens;
After the position of the aberration correction lens is adjusted in the aberration correction step, an adjustment value of the aberration correction lens that maximizes the contrast value of the sample image observed through the immersion objective lens is obtained, and the adjustment is performed. A cover glass thickness detection step for determining the thickness of the cover glass based on the value and the temperature of the immersion liquid measured in the temperature measurement step;
A control step of adjusting the aberration correction lens based on the thickness of the cover glass obtained in the cover glass thickness detection step;
An aberration correction control method comprising: In an aberration correction control method for controlling aberration correction of a microscope including an immersion objective lens including an aberration correction lens,
A temperature measurement step for measuring the temperature of the liquid for immersion filled between the immersion objective lens and the cover glass;
An aberration correction step of adjusting the position of the aberration correction lens based on adjustment value data of the aberration correction lens for correcting aberration in the combination of the temperature of the liquid and the thickness of the cover glass;
In a state where the thickness of the cover glass is unknown, the position of the aberration correction lens based on the adjustment value data corresponding to a combination of different thicknesses of the cover glass with respect to the temperature of the liquid measured by the temperature measurement step. After the control, a combination of thicknesses of the cover glass that maximizes the contrast value of the specimen image observed through the immersion objective lens is obtained, and the aberration correction lens is placed at a position corresponding to the thickness. Control steps to adjust and
An aberration correction control method comprising:

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