JP2011221294A - Apparatus, method and program for focus correction, and microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus, method and program for focus correction, and microscope which can stabilize the focus even when non-optical system elements generate a drift in different direction from the direction of a drift caused by optical system.SOLUTION: The temperature is measured using temperature-sensitive element placed on one side, a plane for a preparation to be set as a reference, and then the temperature is measured using temperature-sensitive element placed on the other side, a plane for a preparation to be set as a reference. The displacement to the focus position is calculated based on the measured temperatures.

Description

  The present invention relates to a focus correction apparatus, a focus correction method, a focus correction program, and a microscope, and is suitable for observing a sample.

  In general, a focus shift called a focus drift occurs due to a temperature change in the surrounding environment or the like. As a technique related to this focus drift, for example, there is Patent Document 1 below.

  In the focus maintaining apparatus in Patent Document 1, the focus detection optical system and the observation optical system are configured by the same member. Therefore, both optical systems are deformed approximately uniformly even when a temperature change occurs, so that the focus on the sample is stably corrected regardless of the deformation.

JP-A-2005-107302

  However, although a drift occurs in the optical axis direction due to the deformation of the optical system, the drift may occur in a direction opposite to the optical axis direction. In this case, the thermal deformation of the stage on which the preparation is arranged is mainly a factor.

  Therefore, in the focus correction apparatus of Patent Document 1, there is a problem that the focus may be corrected at an incorrect position due to a drift that occurs in a direction different from the drift direction of the optical system.

  The present invention has been made in consideration of the above points, and a focus correction apparatus and a focus correction method capable of stabilizing the focus even if there is an element other than the optical system that causes a drift in a direction different from the drift direction of the optical system. A focus correction program and a microscope are proposed.

  In order to solve such a problem, the present invention is a focus correction apparatus, a first measurement unit that measures a temperature using a temperature-sensitive element disposed on one side with respect to a surface on which a preparation is disposed, The second measurement unit that measures the temperature using the temperature-sensitive element disposed on the other side with respect to the surface on which the preparation is disposed, and the temperature measured by the first measurement unit and the second measurement unit. And a calculation unit for calculating a shift amount with respect to the focus position.

  The present invention is also a focus correction method, wherein the first measurement unit measures the temperature using a temperature-sensitive element disposed on one side with respect to the surface on which the preparation is disposed, Measuring the temperature using a temperature-sensitive element disposed on the other side with respect to the surface on which the preparation is to be disposed, and the calculating unit includes the first measuring unit and the second measuring unit. And calculating a deviation amount with respect to the focus position using the measured temperature.

  The present invention is also a focus correction program for measuring a temperature using a temperature-sensitive element disposed on one side with respect to a surface on which a preparation is to be disposed, and the preparation is disposed on a computer. The temperature is measured using a temperature sensing element arranged on the other side with respect to the power plane, and the shift amount with respect to the focus position is calculated using the temperature measured on one side and the other side.

  Further, the present invention is a microscope having a surface on which a preparation is to be arranged, a stage movable in the optical axis direction, and a temperature sensing element arranged on one side with respect to the surface on which the preparation is to be arranged. A first measuring unit that measures the temperature using a temperature measuring element that is disposed on the other side with respect to the surface on which the preparation is to be disposed, and a first measuring unit. And a calculation unit that calculates a deviation amount with respect to the focus position using the temperatures measured by the second measurement unit and the second measurement unit.

  In the present invention, even if the focus drift with respect to the optical axis direction is contradictory on one side and the other side with respect to the preparation arrangement surface, the deviation amount in the optical axis direction with respect to the focus position is calculated from the temperature measured on both sides. The

  As a result, the present invention can calculate the focus drift amount due to the optical system and other elements compared to the case where the drift amount due to the temperature change of the optical system alone is calculated, and the accurate defocus amount. Can be obtained.

It is the schematic which shows the structure of a microscope. It is the schematic where it uses for description of allocation of the imaging | photography range with respect to a biological sample. It is the schematic which shows the structure of a focus correction | amendment part. It is the schematic which shows the functional structure of CPU based on a focus correction program. It is a graph which shows focus drift amount (1) according to temperature change. It is a graph which shows focus drift amount (2) according to temperature change. It is the schematic where it uses for description of the displacement of a focus drift. It is a graph which shows the amount of focus drift according to the temperature change in the case where focus correction is performed, and the case where it is not performed. It is a flowchart which shows a focus correction processing procedure.

Hereinafter, modes for carrying out the invention will be described. The description will be in the following order.
<1. Embodiment>
[1-1. Microscope configuration]
[1-2. Functional configuration of focus correction unit]
[1-3. Specific contents of focus correction processing]
[1-4. Focus correction processing procedure]
[1-5. Effect]
<2. Other embodiments>

<1. Embodiment>
[1-1. Microscope configuration]
In FIG. 1, the structure of the microscope 1 which is this one Embodiment is shown. The microscope 1 has a stage 11 on which a preparation PRT can be placed (hereinafter also referred to as a preparation stage) 11.

  The preparation PRT is obtained by fixing a sample (hereinafter also referred to as a biological sample) in a connective tissue such as blood, epithelial tissue, or both tissues to a slide glass by a predetermined fixing method. Is dyed as necessary. Staining includes not only general staining such as HE (hematoxylin and eosin) staining, Giemsa staining or Papanicolaou staining, but also special staining such as FISH (Fluorescence In-Situ Hybridization) and enzyme antibody method. Is included.

  A light source 12 is disposed on the back side of the preparation stage 11 on which the preparation PRT is to be disposed (hereinafter also referred to as a preparation arrangement surface), and a condenser lens 13 is provided between the preparation stage 11 and the light source 12. Is arranged. The condenser lens 13 collects the light emitted from the light source 12 in the preparation arrangement surface through the through hole HL formed at a predetermined position on the preparation arrangement surface.

  On the other hand, a lens barrel 14 is arranged on the preparation arrangement surface side of the preparation stage 11, and an objective lens 15 is arranged on one end of the lens barrel 14 facing the preparation arrangement surface. An imaging element 16 is disposed at the other end of the lens barrel 14, and an image reflected on the lens surface of the objective lens 15 is formed on the imaging surface of the imaging element 16.

  On the other hand, a stage drive mechanism 17 is connected to the preparation stage 11. The stage driving mechanism 17 is a mechanism that can individually drive the preparation stage 11 in a direction (Z direction) orthogonal to a direction (X direction, Y direction) parallel to the preparation arrangement surface.

  Specifically, as shown in FIG. 2, the preparation stage 11 is scanned in a direction parallel to the preparation arrangement surface so that the imaging range PR is assigned to the biological sample SPL arranged on the preparation PRT.

  Further, every time the imaging range PR to be assigned to the biological sample is changed, the optical axis LX orthogonal to the preparation arrangement surface is focused so that the objective lens 15 is focused on the focus position detected with respect to the imaging range PR. The preparation stage 11 is adjusted in the direction.

  In the case of this embodiment, the focus position is detected by a focus detection unit 18 connected to the image sensor 16. For example, the focus detection unit 18 changes the depth position in the imaging range PR at predetermined intervals, and determines the depth position where the image contrast at the depth position is the largest as the focus position.

  An image processing unit 19 is also connected to the image sensor 16. The image processing unit 19 performs various types of image processing on the subject image data output from the image sensor 16.

  In the image processing, for example, an image of a sample portion existing within an imaging range PR assigned to a biological sample is connected using a predetermined connection algorithm, and the biological sample image (enlarged by the magnification of the objective lens 14). There is a process for generating an image of the entire biological sample.

  Further, when the biological sample image is generated, the image processing unit 19 stores the biological sample image in a storage medium in units of subjects.

  In addition to this configuration, the microscope 1 is provided with a focus correction unit 20 that corrects the focus position according to the temperature.

[1-2. Configuration of focus correction unit]
As shown in FIG. 3, the focus correction unit 20 is configured by connecting various hardware to a CPU (Central Processing Unit) 41.

  Specifically, for example, a ROM (Read Only Memory) 22, a RAM (Random Access Memory) 23 as a work memory of the CPU 21, an operation input unit 24 for inputting a command according to a user operation, an interface 25, a display unit 26, and a memory The unit 27 is connected via the bus 28.

  The ROM 22 stores programs for executing various processes. A stage driving mechanism 17, a focus detection unit 18, and an image processing unit 19 are connected to the interface 25.

  A liquid crystal display, an EL (Electro Luminescence) display, a plasma display, or the like is applied to the display unit 26. For the storage unit 27, a magnetic disk represented by HD (Hard Disk), a semiconductor memory, an optical disk, or the like is applied. A portable memory such as a USB (Universal Serial Bus) memory or a CF (Compact Flash) memory may be applied.

  The CPU 21 develops, in the RAM 23, a program associated with an instruction given from the operation input unit 24 or the like among a plurality of programs stored in the ROM 22. Further, the CPU 21 appropriately controls the display unit 26 or the storage unit 27 in accordance with the program developed in the RAM 23.

  In addition, the CPU 41 exchanges data with the stage drive mechanism 17, the focus detection unit 18, or the image processing unit 19 as appropriate through the interface 45 in accordance with a program developed in the RAM 23.

[1-3. Specific contents of focus correction processing]
For example, when a start instruction to search for in-focus is given, the CPU 21 develops a focus correction program associated with the instruction in the RAM 23. The CPU 21 functions as a stage temperature measuring unit 31, a lens barrel temperature measuring unit 32, a drift amount calculating unit 33, and a defocus amount calculating unit 34 as shown in FIG. 4 according to the focus correction program.

  The stage temperature measurement unit 31 measures the temperature at predetermined intervals using a temperature sensitive element arranged on the surface of the preparation stage 11 and sends the measurement result as data to the drift amount calculation unit 33.

  The lens barrel temperature measuring unit 32 measures the temperature at predetermined intervals using a temperature sensing element arranged inside the lens barrel 14 and sends the measurement result as data to the drift amount calculating unit 33.

  The drift amount calculation unit 33 recognizes the focus position detected by the focus detection unit 18 and uses the stage temperature measurement unit 31 and the shift amount in the optical axis direction with respect to the focus position (hereinafter also referred to as a focus drift amount). Calculation is performed using the measurement result of the lens barrel temperature measurement unit 32.

  Specifically, the temperature measured by the stage temperature measurement unit 31 is t1, the coefficient for the temperature t1 is k1, the focus drift amount is D, the temperature measured by the lens barrel temperature measurement unit 32 is t2, When the coefficient for the temperature t2 is k2 and the constant is d,

  D = d + (k1 × t1) + (k2 × t2) (1)

Accordingly, the focus drift amount is calculated. The units of the coefficients k1 and k2 are [μm / ° C.], the coefficient k1 is a positive value, and the coefficient k2 is a negative value.

  In general, the focus drift amount varies according to the temperature change. Here, as a result of the experiment, a graph of the focus drift amount according to the temperature change is shown in FIG. 5, and a graph in which the right side and the left side are overlapped with the three-time axis in the lower graph of FIG. It is shown in FIG. As can be seen from these graphs, there is a portion where the response speed to temperature is faster than the others (portion surrounded by a broken line), and it is not a simple fluctuation proportional to the temperature change.

  The main factor is that the focus drift in the direction of the optical axis LX is contradictory on one side (lower side) and the other side (upper side) with respect to the preparation arrangement surface to be focused, and the speed at which the focus drift occurs is Because it is different.

  A part that causes a focus drift on one side (lower side) with respect to the preparation arrangement surface is mainly the preparation stage 11. In addition, a component that causes a focus drift on the other side (upper side) is a structure (a lens barrel 14 including the objective lens 15 and the image sensor 16) that mainly constitutes an optical system.

  It may be important to drive the preparation stage 11 at a high speed, and the preparation stage 11 generally has a smaller heat capacity and volume than the structure constituting the optical system. For this reason, as shown in FIG. 7, when the temperature rises, the preparation stage 11 responds faster than the structure constituting the optical system and thermally expands, and the focus plane drifts upward due to this (see FIG. 7). FIG. 7 (A)).

  As the temperature rises further, the thermal expansion of the preparation stage 11 begins to settle, while the thermal expansion of the structure constituting the optical system starts and gradually increases, and the focus surface drifts downward (FIG. 7B). )).

  When the temperature falls, the preparation stage 11 first contracts and the focus surface drifts downward. When the temperature further falls, the preparation stage 11 starts to contract, while the structure constituting the optical system starts to contract. The focus surface gradually drifts upward.

  By the way, the material and size of the preparation stage 11 vary depending on the type of product, and the number and structure of the parts attached to the preparation stage 11 are various. On the other hand, the material and size of the structure constituting the optical system, the number of lenses, and the like vary depending on the type of product, and the number and structure of components attached to the structure vary.

  For this reason, the response speed of the focus drift with respect to the temperature is uncertain, but a representative factor that affects the response speed can be specified. Therefore, the coefficients k1 and k2 and the constant d in the equation (1) can be set by associating with an identifier such as a product model number. For the same type of product, even if the coefficients k1 and k2 and the constant d are fixed, there is no substantial difference in the calculated focus drift amount.

  Also, the fact that the focus drifts that occur in the optical axis LX direction on one side (lower side) and the other side (upper side) with respect to the preparation arrangement surface that is the focus target is contradictory, even if the type of product is different. Absent. The preparation stage 11 represented as one side (lower side) and the lens barrel 14 represented as the other side (upper side) exist in any product.

  Therefore, the optical system and the optical system can be obtained by a simple product-sum operation of adding the result of multiplying the surface temperature t1 of the preparation stage 11 by the positive coefficient k1 and the result of multiplying the internal temperature t2 of the lens barrel 14 by the negative coefficient k2. It is possible to calculate the focus drift amount due to other elements.

  When the drift amount calculation unit 33 calculates the focus drift amount, the defocus amount calculation unit 34 corrects the focus position that is a reference for the focus drift amount using the focus drift amount. The defocus amount calculation unit 34 calculates the amount of movement of the preparation stage 11 to the corrected focus position (hereinafter also referred to as a defocus amount), and supplies this to the stage drive mechanism 17.

  Here, FIG. 8 shows a focus drift amount corresponding to a change in temperature when the focus correction is executed and when the focus correction is not executed. In this experiment, the coefficient k1 was set to 4.3125 [μm / ° C.], and the coefficient k2 was set to −8.6106 [μm / ° C.]. As shown in FIG. 8, the fluctuation range of the focus drift amount is greatly reduced as compared with the case of not executing.

  In the experimental results shown in FIG. 8, the focus drift amount when correction is performed in the vicinity of the third hour on the time axis exceeds −2 μm, but this is due to rapid cooling. However, in reality, it is difficult to consider use in an environment where the temperature changes rapidly in a short time as in this experiment. When focus correction is performed under an environment that can normally be assumed, it is considered that the focus drift amount corresponding to the temperature change can be suppressed to approximately ± 1 [μm] or less.

[1-4. Focus correction processing procedure]
Next, the focus correction processing procedure in the CPU 21 will be described with reference to the flowchart shown in FIG.

  For example, when the CPU 21 receives a start instruction to search for in-focus, the CPU 21 starts the focus correction processing procedure and proceeds to the first step SP1. In the first step SP1, the CPU 21 acquires parameters (coefficients k1, k2 and a constant d) stored in the storage unit 47 as setting values required for calculating a deviation amount (focus drift amount) with respect to the focus position, and the second step Proceed to SP2.

  In the second step SP2, the CPU 21 waits for data indicating that the focus position in the shooting range PR has been detected. When the data is received from the focus detection unit 18, the CPU 21 proceeds to the third step SP3.

  In the third step SP3, the CPU 21 acquires data indicating the focus position from the focus detection unit 18, and proceeds to the fourth step SP4.

  In the fourth step SP4, the CPU 21 measures the surface temperature of the preparation stage 11 and the internal temperature of the lens barrel 14, and proceeds to the fifth step SP5. In the fifth step SP5, the CPU 21 calculates the focus drift amount by the equation (1) using the temperature measured in the fourth step SP4 and the parameter acquired in the first step SP1, and the CPU 21 calculates the value corresponding to the calculation result. A focus amount is given to the stage drive mechanism 17.

  Then, the CPU 21 proceeds to the sixth step SP6, determines whether or not a specified interval has passed since the previous measurement time, and returns to the fourth step SP4 when the specified interval has passed. On the other hand, if the specified interval has not yet passed, the CPU 21 proceeds to the seventh step SP7 and determines whether or not data indicating that the image in the current shooting range PR has been received has been received.

  In this manner, the CPU 21 adjusts the focus position by continuously giving the defocus amount corresponding to the focus drift amount to the stage driving mechanism 17 at every predetermined interval until the image in the current shooting range PR is stored.

  On the other hand, when the CPU 21 receives data indicating that the image in the current shooting range PR is stored from the image processing unit 19, the CPU 21 proceeds to the eighth step SP8. In the eighth step SP8, the CPU 21 waits for data indicating that storage of an image of a new imaging range PR is started or data indicating that all images assigned to the biological sample SPL have been stored.

  Here, when data indicating that storage of an image of a new photographing range PR is started is given from the image processing unit 19, the CPU 21 returns to the second step SP2 and repeats the above-described processing.

  On the other hand, when data indicating that all the images assigned to the biological sample SPL have been stored is given from the image processing unit 19, the CPU 21 ends the focus correction processing procedure.

[1-5. Effect]
In the above configuration, the microscope 1 measures the surface temperature of the preparation stage 11 which is a main factor of focus drift on one side (lower side) with the preparation arrangement surface as a reference. The microscope 1 also measures the internal temperature of the structure (the lens barrel 14 including the objective lens 15 and the imaging element 16) that constitutes the optical system that is a main factor of focus drift on the other side (upper side) with respect to the preparation arrangement surface. taking measurement.

  Then, the microscope 1 calculates a deviation amount in the optical axis direction with respect to the focus position using these measurement results.

  Therefore, in this microscope 1, even if the focus drift in the optical axis LX direction is contradictory on one side (lower side) and the other side (upper side) with respect to the preparation arrangement surface, the focus position is determined from the temperature measured on both sides. The amount of deviation in the optical axis direction with respect to is calculated.

  As a result, the microscope 1 can calculate the focus drift amount due to the optical system and other elements compared to the case where the drift amount due to the temperature change of the optical system alone is calculated, and the accurate defocus can be calculated. An amount can be obtained.

  By the way, it may be important to drive the preparation stage 11 at high speed. In general, the preparation stage 11 has a smaller heat capacity and volume than the structure constituting the optical system. Therefore, when the focus drifts in the direction of the optical axis LX are contradictory, the speed at which the focus drift occurs is different and does not result in a simple fluctuation proportional to the temperature change (FIGS. 5 and 6).

  However, in this microscope 1, the amount of deviation in the optical axis direction with respect to the focus position is negative for the temperature measured on one side (lower side) multiplied by a positive coefficient and the temperature measured on the other side (upper side). It is calculated by adding the result obtained by multiplying the coefficient. For this reason, even when the drift speeds contradictory to each other in the optical axis LX direction are different and the fluctuation is not proportional to the temperature change, the focus drift amount caused by the optical system and other elements can be calculated. Become.

  Further, in this microscope 1, the part whose temperature is to be measured on one side (lower side) is the surface of the preparation stage 11 that becomes the main factor of focus drift on the one side. The part whose temperature is to be measured on the other side (lower side) is in the lens barrel 14 which is the main factor of focus drift on the other side.

  Therefore, in this microscope 1, the identification of the coefficient may be simple and clear, and the optical system and other parts are more accurately compared to the case where the side temperature place is not near the preparation stage 11 and the inside of the lens barrel 14. The amount of focus drift due to the element can be calculated.

  According to the above configuration, the microscope 1 that can stabilize the focus even if there is an element other than the optical system that causes drift in a direction different from the drift direction of the optical system is realized.

<2. Other embodiments>
In the above-described embodiment, the tissue section is a biological sample. However, the biological sample is not limited to this embodiment. For example, smear cells or chromosomes can be applied as the biological sample. Further, for example, a sample other than a living body (organism) such as a semiconductor element may be used.

  In the above-described embodiment, the temperature of the surface of the preparation stage 11 is measured using the temperature sensitive element arranged on the surface of the preparation stage 11. However, the place where the temperature sensitive element is to be arranged is not limited to the surface of the preparation stage 11. For example, it may be arranged on the surface of a member that supports the preparation stage 11 or a member that positions the preparation PRT at a predetermined position on the preparation arrangement surface. In short, it may be arranged near the preparation stage 11. In addition, the number (location to be measured) where the temperature sensitive element should be arranged may be plural.

  In the above-described embodiment, the temperature in the lens barrel is measured using the temperature sensitive element arranged inside the lens barrel 14. However, the place where the temperature sensitive element is to be arranged is not limited to the inside of the lens barrel 14. For example, all or part of the lens barrel 14 may be a temperature sensitive element. In addition, the number (location to be measured) where the temperature sensitive element should be arranged may be plural.

  In the above-described embodiment, the image sensor for obtaining an image for photographing and the image sensor for obtaining an image for focus search are shared. However, an image sensor for obtaining an image for photographing and an image sensor for obtaining an image for focus search may be provided separately.

  In this embodiment, the temperature measured for the structure constituting the optical system that forms an image on the image sensor for obtaining the image for focus search is t2, and the focus drift amount is calculated from the equation (1). If calculated, the focus can be stabilized. In addition to this, the focus can be further stabilized by considering the temperature of the structure constituting the optical system that forms an image on the image sensor for obtaining an image for photographing. Specifically, if the temperature to be considered is t3 and the negative coefficient for the temperature t3 is k3,

  D = d + (k1 * t1) + (k2 * t2) + (k3 * t3) (2)

It becomes.

  Further, in the above-described embodiment, the focus position is determined as the depth position where the depth position in the imaging range PR is changed at predetermined intervals and the image contrast at the depth position is the largest. However, the method for determining the focus position is not limited to this embodiment. For example, an image sensor for focus search separately from an image sensor for photographing, and an optical system that forms two subject images with different viewpoints (hereinafter also referred to as phase difference images) on the image sensor Can be determined from the parallax of the phase difference image. Of course, other determination methods may be used.

  In the above-described embodiment, an image of light that passes through the sample is formed, but an image of light that reflects the sample may be formed.

  In the embodiment described above, the present invention is applied to the microscope 1. However, the apparatus to which the present invention is applied is not limited to the microscope 1. The present invention can be applied to various apparatuses having a stage on which a sample is arranged and movable in the optical axis direction.

  It should be noted that various forms can be widely adopted without departing from the spirit of the present invention, even if other than the matters described as other embodiments.

  The present invention can be used in the bio-industry such as genetic experiments, creation of medicines, or patient follow-up.

  DESCRIPTION OF SYMBOLS 1 ... Microscope, 11 ... Preparation stage, 12 ... Light source, 13 ... Condenser lens, 14 ... Lens barrel, 15 ... Objective lens, 16 ... Imaging element, 17 ... Stage drive mechanism, 18 ... Focus detection unit, 20 ... focus correction unit, 31 ... stage temperature measurement unit, 32 ... lens barrel temperature measurement unit, 33 ... drift amount calculation unit, 34 ... defocus amount calculation unit, LX ... optical axis , PR: Shooting range, PRT: Preparation, SPL: Biological sample.

Claims (7)

A first measurement unit that measures a temperature using a temperature-sensitive element disposed on one side with respect to the surface on which the preparation is to be disposed;
A second measuring unit that measures the temperature using a temperature sensing element disposed on the other side with respect to the surface on which the preparation is to be disposed;
A focus correction apparatus comprising: a calculation unit that calculates a deviation amount with respect to a focus position using the temperatures measured by the first measurement unit and the second measurement unit. The calculation unit is
The result obtained by multiplying the temperature measured by the first measurement unit by a positive coefficient and the result obtained by multiplying the temperature measured by the second measurement unit by a negative coefficient are added. Focus correction device. The first measurement unit includes:
Measure the temperature near the stage with the surface on which the preparation is to be placed,
The second measuring unit is
The focus correction apparatus according to claim 2, wherein the temperature in the lens barrel is measured. The first measurement unit includes:
Measure the temperature using a temperature sensing element placed near the stage with the surface where the preparation should be placed,
The second measuring unit is
The focus correction apparatus according to claim 2, wherein the temperature is measured using a temperature sensitive element arranged in the lens barrel. A step of measuring a temperature using a temperature sensing element disposed on one side with respect to a surface on which a preparation is to be disposed;
A step of measuring a temperature using a temperature sensing element disposed on the other side with respect to a surface on which the preparation is to be disposed,
A calculation unit having a step of calculating a deviation amount with respect to a focus position using temperatures measured by the first measurement unit and the second measurement unit. Against the computer,
Measuring the temperature using a temperature sensitive element arranged on one side with respect to the surface on which the preparation is to be arranged,
Measuring the temperature using a temperature sensitive element disposed on the other side with respect to the surface on which the preparation is to be disposed;
A focus correction program for executing a calculation of a deviation amount with respect to a focus position using temperatures measured on one side and the other side. A stage having a surface on which the preparation should be arranged and movable in the optical axis direction;
A first measurement unit that measures temperature using a temperature-sensitive element disposed on one side with respect to the surface on which the preparation is to be disposed;
A second measuring unit that measures the temperature using a temperature sensing element disposed on the other side with respect to the surface on which the preparation is to be disposed;
A microscope having a calculation unit that calculates a deviation amount with respect to a focus position using temperatures measured by the first measurement unit and the second measurement unit.