JP2013120285A - Microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microscope device capable of obtaining a three dimensional stereoscopic image of the whole observation area of a sample while suppressing increase in size of the whole device, increase in image data volume, and increase in imaging time, even when deviation of focal point of the objective lens occurs.SOLUTION: A microscope device 1 includes an objective lens 4 focusing on a sample 3, an imaging element 12 for imaging the sample 3 focused by the objective lens 4, and a stage 5 capable of moving while holding the sample 3. The microscope device moves the stage 5 in an optical axis direction of the objective lens 4 and images the sample 3 at a plurality of different positions in the optical axis direction so as to acquire a plurality of images of the sample 3. The microscope device 1 further includes image identification means 17 for identifying a sample image containing the image of the sample 3 and a non-sample image not containing the images of the sample 3 from among the plurality of images, and non-sample image recording means 18 for acquiring variation of the number of the non-sample images. Based on the variation, the microscope device 1 adjusts an imaging starting point of the stage 5.

Description

  The present invention relates to a microscope apparatus that acquires a three-dimensional stereoscopic image of a sample.

  Conventionally, when observing a sample (cell, tissue section, etc.) using a microscope device, there has been a method (Z stack) for continuously acquiring slice images of a cross section of the sample at a plurality of different positions in the optical axis direction of the objective lens. Are known. In the Z stack, a plurality of slice images are acquired by shifting the stage holding the sample in the optical axis direction at a constant sampling pitch. According to this Z stack, a three-dimensional stereoscopic image of a sample can be formed by synthesizing a plurality of slice images of a portion (observation region) to be observed in the sample by image processing.

  However, when the focus position fluctuates due to, for example, changes in the refractive index of the optical element in the objective lens or thermal deformation of the lens barrel holding the optical element due to temperature fluctuations in the surrounding environment, heat generation inside the apparatus, etc. There is. In general, the temperature sensitivity of the objective lens is high, and the focal position shifts by about 10 μm when the environmental temperature fluctuates by about 5 ° C. When defocusing occurs due to fluctuations in the focal position, a part of the observation area of the sample does not fit within the set imaging range in the optical axis direction, and a part of the image to be acquired is missing, resulting in a three-dimensional stereoscopic image of the entire observation area Cannot be formed. For this reason, in the microscope apparatus, it is important to suppress the influence of temperature fluctuations and to maintain the focal position stably.

  Here, Patent Document 1 proposes a three-dimensional microscope system including a focus error detection optical system that detects a shift in the focal position of the objective lens caused by thermal deformation and outputs a focus error signal corresponding to the shift. ing. In this system, the deviation of the focus position is corrected by controlling the actuator based on the focus error signal. Further, Patent Document 2 proposes a scanning confocal microscope apparatus including a member for enclosing each member such as an objective lens and a confocal pinhole with an exterior cover made of a heat insulating material and isolating the internal space. Yes. In this apparatus, the temperature of the air inside the exterior cover is adjusted by a temperature sensor and a heater, thereby preventing distortion of the optical system and the mechanical system due to the thermal effect.

JP 2008-122629 A JP 2008-256927 A

  However, the configuration described in Patent Document 1 requires a space for newly providing a focus error detection optical system, and the focus error detection optical system itself may be thermally deformed. Further, in the configuration described in Patent Document 2, when high-precision temperature management for the objective lens is required, a large amount of temperature-controlled air is required, which leads to an increase in the size and cost of the entire apparatus.

  Here, taking into account the amount of focal position deviation caused by temperature fluctuations in the surrounding environment, a preliminary imaging range is provided in the optical axis direction of the objective lens to increase the stage driving amount, thereby widening the measurement range of the sample. A method is conceivable. However, since the amount of deviation of the focal position of the objective lens due to temperature fluctuation or the like is large with respect to the thickness of the sample in the optical axis direction, it is necessary to provide a large number of preliminary imaging ranges. Therefore, in this method, the number of slice images with respect to the preliminary imaging range is very large as compared with slice images necessary for observation, and the amount of image data and imaging time increase.

  Therefore, the present invention acquires a three-dimensional stereoscopic image of the entire observation region of the sample while suppressing an increase in the size of the entire apparatus and an increase in the amount of image data and imaging time even when the focal position of the objective lens shifts. An object of the present invention is to provide a microscope apparatus capable of performing the above.

  In order to achieve the above object, a microscope apparatus according to one aspect of the present invention includes an objective lens that forms an image of a sample, an imaging element that images the sample imaged by the objective lens, and the sample. And moving the stage in the optical axis direction of the objective lens, and imaging the sample at a plurality of positions different in the optical axis direction, thereby obtaining a plurality of images of the sample. A microscope apparatus for obtaining the image recognition means for identifying a sample image including the image of the sample and a non-sample image not including the image of the sample, and the number of the non-sample images. And a non-sample image recording means for acquiring the fluctuation amount of the stage, and adjusting the imaging start position of the stage based on the fluctuation amount.

  Further objects and other features of the present invention will become apparent from the preferred embodiments described below with reference to the accompanying drawings.

  According to the present invention, even when the focal position of the objective lens shifts, a three-dimensional stereoscopic image of the entire observation region of the sample is suppressed while suppressing an increase in the size of the entire apparatus and an increase in the amount of image data and imaging time. A microscope apparatus that can be obtained can be provided.

1 is a block diagram showing a microscope apparatus according to Embodiment 1 of the present invention. Explanatory drawing which shows the adjustment operation of the stage in the microscope apparatus which concerns on Example 1 of this invention. FIG. 6 is a block diagram showing a microscope apparatus according to Embodiment 2 of the present invention. FIG. 6 is a block diagram showing a microscope apparatus according to Embodiment 3 of the present invention. FIG. 9 is a block diagram showing a microscope apparatus according to Embodiment 4 of the present invention.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

[Example 1]
With reference to the block diagram shown in FIG. 1, the operation of imaging the sample 3 to be observed by the microscope apparatus 1 according to the first embodiment of the present invention will be described. The sample 3 is disposed and fixed on a stage 5 provided with an opening, and light from a light source (not shown) is irradiated to the sample 3 through the opening. The light 6 that has passed through the sample 3 is imaged on the imaging surface of the image sensor 12 via the objective lens 4, and the image of the sample 3 is converted into an electrical signal by the image sensor 12. Then, this electric signal is sent to the image processing means 15 and subjected to image processing to form an image. Note that the imaging operation by the imaging device 12 can be controlled by the imaging device control means 13.

  In this embodiment, a slice image of the sample 3 is acquired by using a method (Z stack) in which the above-described imaging operation is performed at a plurality of positions in the Z direction (the optical axis direction of the objective lens 4) in the sample. Specifically, the stage controller 14 step-drives the stage 5 in the Z direction, whereby the sample 3 can be imaged at a plurality of different positions in the Z direction to obtain slice images. Then, the acquired slice images can be synthesized by the image processing means 15 to form a three-dimensional stereoscopic image of the entire sample 3. Each acquired slice image and three-dimensional stereoscopic image are stored in the image database 16 and can be displayed on a PC monitor (not shown). In addition, when performing such Z stacking, it is desirable to use a confocal microscope as the microscope apparatus 1.

  Here, a change in the refractive index of the optical element in the objective lens 4 or a thermal deformation of the lens barrel holding the optical element may occur due to a temperature change in the surrounding environment, and the focal position may change. Therefore, in the present embodiment, a preliminary imaging range is provided in the Z direction in consideration of the amount of focus position shift caused by temperature fluctuation or the like. That is, by setting a preliminary stroke in the step drive of the stage 5, the measurement range in the Z direction of the sample 3 is expanded. This eliminates the need for an optical system for detecting the shift amount of the focal position and a configuration for adjusting the temperature of the objective lens 4, so that the entire observation area of the sample is not increased without increasing the size of the entire apparatus. A three-dimensional stereoscopic image can be acquired.

  However, even in a room whose temperature is controlled by an air conditioner such as an air conditioner, a temperature fluctuation of about 2 to 3 ° C. generally occurs. The amount of shift of the focal position of the objective lens 4 due to this temperature fluctuation is the thickness of the sample 3 It is big against. Therefore, it is necessary to provide a large number of preliminary imaging ranges according to the focal position shift amount. For example, assuming that the focal position shift amount of the objective lens 4 when a temperature fluctuation of 3 ° C. occurs is 7.5 μm, slice images of cells having a thickness of 5 μm are acquired at a sampling pitch of 0.5 μm at regular intervals. Consider the case. In this case, in order to obtain a three-dimensional stereoscopic image of the entire cell, eleven slice images are required for a cell thickness of 5 μm. On the other hand, when a preliminary stroke corresponding to a focal position shift amount of 7.5 μm is set for the front surface side and the back surface side of the cell, the number of preliminary captured images of slice images obtained by imaging with the preliminary stroke is 30. It becomes a sheet. Therefore, this method has a problem that the number of slice images for the preliminary imaging range is very large as compared with slice images necessary for observation, and the amount of image data and imaging time increase.

  Therefore, in the microscope apparatus 1, in the case where the preliminary imaging range is provided in consideration of the shift amount of the focus position caused by the temperature fluctuation of the surrounding environment, a configuration for reducing the number of preliminary imaging and shortening the imaging time will be described below. To do.

  As shown in FIG. 1, the microscope apparatus 1 includes a condition setting unit 2, an image recognition unit 17, a non-sample image recording unit 18, and a correction amount calculation unit 19 in addition to the configuration described above. . Note that a CPU can be used as each of these units, the image sensor control unit 13 and the image processing unit 15. In addition to the CPU, the stage control means 14 may include an actuator for driving the stage and a stage position detector (for example, an encoder).

  The initial setting when performing Z stacking is performed by the condition setting means 2 as follows. First, based on the thickness information of the sample 3 acquired in advance, as shown in FIG. 2A, a sample stroke 7 that is a stage movement amount for sample imaging is set. The number of sampled images is obtained from the sample stroke 7 and the sampling pitch arbitrarily set by the user. Also, preliminary strokes 8a and 8b are set in addition to the sample stroke 7, and the number of preliminary images is obtained from the sampling pitch in the same manner as the number of sample images. Here, in FIG. 2A, the preparatory strokes 8a and 8b are set on the upper side and the lower side of the sample stroke 7, and are included in the imaging stroke 9. Then, the condition setting means 2 determines the imaging start position of the stage 5 in order to set the imaging range for imaging according to the imaging stroke 9, and calculates the number of imaging from the sample imaging number and the preliminary imaging number. In this way, the initial setting information obtained by the condition setting unit 2 is sent to the stage control unit 14 and the image sensor control unit 13, whereby imaging of the sample 3 is started.

  A plurality of slice images acquired based on each setting information described above are stored in the image database 16. Here, the image recognition means 17 discriminates between the sample image including the image of the sample 3 and the non-sample image not including the image of the sample 3 based on the acquired contrast of each slice image. At this time, slice images (front surface image and back surface image) at the front surface position and the back surface position of the sample 3 can be detected. Then, the non-sample image recording unit 18 acquires and records the number of non-sample images on the front side and the back side of the sample 3 based on the identification result of the image recognition unit 17. The non-sample image recording means 18 records the number of non-sample images as needed every time a three-dimensional stereoscopic image is acquired, such as when observing the same sample 3 a plurality of times or continuously observing a plurality of samples 3. Go. Since the front image and the back image are images of the boundary where the sample image is switched to the non-sample image, if the front image and the back image are detected by the image recognition means 17, the number of non-sample images can be easily detected. can do.

  Here, in FIG. 2B, the position where the uppermost end of the imaging range coincides with the focal position of the objective lens 4 is set as the imaging start position of the stage 5, and the deviation of the focal position of the objective lens 4 occurs. It shows a case that is not. Here, it is assumed that the imaging is performed in the order from the front surface side to the back surface side of the sample 3, but the imaging start position of the stage 5 may be set so that the imaging is performed in the order from the back surface side to the front surface side of the sample 3. Good. In addition, in the imaging range captured with respect to the imaging stroke 9, the range in which the sample 3 is imaged is defined as the sample imaging range 10, and the range in which the sample 3 is not imaged is defined as the non-sample imaging ranges 11a and 11b. Then, since the deviation of the focal position of the objective lens 4 does not occur, the sample stroke 7 set by the condition setting means 2 coincides with the sample imaging range 10. That is, the preliminary strokes 8a and 8b coincide with the non-sample imaging range 11a on the front side of the sample 3 and the non-sample imaging range 11b on the back side, respectively, and the number of non-sample images in the non-sample imaging ranges 11a and 11b is the same. It becomes equal to the number of preliminary images.

  On the other hand, when the sample 3 is continuously observed, a temperature variation or the like of the surrounding environment occurs, and when the focal position of the objective lens 4 is deviated, the non-sample image recorded at any time by the non-sample image recording means 18. The number of sheets will fluctuate. For example, as shown in FIG. 2C, when the focal position moves to the objective lens 4 side, the imaging range set by the condition setting means 2 also moves by the same amount as the focal position deviation amount. Therefore, the non-sample imaging range 11a (the number of non-sample images) on the front side of the sample 3 increases, and the non-sample imaging range 11b (the number of non-sample images) on the back side of the sample 3 decreases.

  Therefore, the non-sample image recording means 18 calculates the variation amount of the number of non-sample images every time a three-dimensional stereoscopic image of the sample 3 is acquired or every time the sample 3 is changed based on the recorded number of non-sample images. Record. At this time, using the number of preliminary images set by the condition setting means 2 as a reference for the number of non-sample images, the amount of variation from that reference is acquired, or the difference in the number of non-sample images recorded for each observation of the sample 3 is acquired. By doing so, the deviation amount 20 of the focal position may be calculated.

  Further, the correction amount calculation means 19 can calculate the focal position deviation amount 20 of the objective lens 4 based on the fluctuation amount of the number of non-sample images. The correction amount calculation means 19 can set in advance an allowable variation amount of the number of non-sample images (allowable number of non-sample images) corresponding to the allowable range of the focal position deviation amount 20. When the number of non-sample images on the front side or the back side of the sample 3 changes by the allowable fluctuation amount and matches the allowable number, it is determined that the imaging start position of the stage 5 needs to be adjusted. . Specifically, the shift amount 20 of the focal position is fed back to the stage control unit 14 as an imaging range correction amount 21 so that the non-sample imaging range 11a and the non-sample imaging range 11b of the sample 3 are equal. Adjust the imaging start position. As a result, the influence of the focal position shift amount 20 is canceled and the imaging range is corrected.

  As a specific example, the thickness of the sample 3 is 5 μm, the sampling pitch of the slice image is 0.5 μm, and the amount of deviation of the focal position of the objective lens 4 when a temperature variation of 3 ° C. occurs is 7.5 μm. Think.

  In this case, the condition setting means 2 can set the sample stroke 7 to 5 μm based on the thickness information of the sample 3, and the number of sampled images calculated at this time is 11. Here, when the present invention is not used, it is necessary to set each of the preliminary strokes 8a and 8b to 7.5 μm in consideration of the focal position deviation amount of 7.5 μm. The number will be 30. However, in the present embodiment, as described above, since the imaging start position of the stage 5 can be adjusted at any time according to the shift of the focal position, each of the preliminary strokes 8a and 8b is set to a small value (for example, 2 μm). can do. When the preliminary strokes 8a and 8b are each 2 μm, the number of preliminary images to be captured is eight (four each), and the number of preliminary images to be captured is reduced as compared with the case where the preliminary strokes are provided without applying the present invention. Can do. When the temperature fluctuations in the surrounding environment are small, the preliminary strokes 8a and 8b can be made smaller.

  When the focal position of the objective lens 4 is not shifted, as shown in FIG. 2B, the number of non-sample images on the front side and the back side of the sample 3 is equal to the set number of pre-imaging images (each 4 Sheet). However, as shown in FIG. 2C, when the focal position of the objective lens 4 is shifted upward with time, the number of non-sample images on the surface side of the sample 3 is 4 to 5, 6 and so on. As the number increases, the number of non-sample images on the back side of the sample 3 decreases from four to three and two. Here, when the allowable variation amount of the number of non-sample images is set to 2 by the correction amount calculation means 19, the allowable number of non-sample images on the front side and the back side of the sample 3 is 6 and 2, respectively. Therefore, when the number of non-sample images acquired by the non-sample image recording means 18 matches this allowable number, it is determined that the imaging range needs to be corrected. Then, the correction amount calculation means 19 calculates the focal position deviation amount 20 of the objective lens 4 as 1 μm based on the sampling pitch (0.5 μm) of the slice image and the variation amount (2 sheets) of the number of non-sample images. Is done. Therefore, the deviation amount 20 is sent to the stage control unit 14 as the imaging range correction amount 21 so that feedback adjustment with respect to the imaging start position of the stage 5 is performed. As a result, the imaging range is corrected with respect to the focal position shift amount 20, and the number of non-sample images on the front surface side and the back surface side of the sample 3 can be returned to 4, respectively.

  As described above, according to the microscope apparatus 1 according to the present embodiment, even when the focus position of the objective lens 4 is deviated, the stage 5 is feedback adjusted based on the correction amount calculated from the change in the number of non-sample images recorded at any time. Thus, the imaging range can be corrected. Therefore, even if the preliminary strokes 8a and 8b are set to be small, a three-dimensional stereoscopic image of the entire observation region of the sample 3 can be acquired, so that the overall size of the apparatus and the increase in image data amount and imaging time are suppressed. can do.

[Example 2]
Hereinafter, another embodiment of the present invention will be described with reference to FIG. In the following description, the same or equivalent components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is simplified or omitted.

  The microscope apparatus 1 according to the present embodiment includes a preliminary imaging adjustment unit 22 in addition to the configuration of the first embodiment. For example, when the amount of temperature fluctuation in the surrounding environment is smaller than originally assumed, the condition setting means 2 uses the focal position shift amount 20 of the objective lens 4 as shown in FIG. An unnecessary portion is generated in the set preliminary portion strokes 8a and 8b. Therefore, the preliminary imaging adjustment unit 22 acquires an unnecessary amount (unnecessary number) of the number of non-sample images from the fluctuation amount of the number of non-sample images acquired by the non-sample image recording unit 18 and corrects the preliminary amount from the unnecessary number. Calculate the value. Then, by feeding back the preliminary correction value to the condition setting unit 2, the preliminary strokes 8a and 8b set by the condition setting unit 2 in the subsequent measurement can be reduced, and the amount of image data and the imaging time are increased. Can be suppressed.

  Here, based on the specific example described in the first embodiment, the preliminary imaging adjustment unit 22 of the present embodiment will be described. For example, when the preliminary imaging adjustment unit 22 detects an unnecessary portion of the number of non-sample images, it is determined that the preliminary strokes 8a and 8b (each 2 μm) are too large with respect to the focal position shift amount 20 of the objective lens 4. Then, the preliminary imaging adjustment unit 22 obtains an unnecessary number (for example, one each) of the preliminary imaging number (four each) corresponding to the preliminary strokes 8a and 8b, and sets the calculated preliminary correction value as the condition setting unit. Feedback to 2. Then, based on this preliminary correction value, the preliminary setting strokes 8a and 8b are newly set by the condition setting means 2, whereby the number of preliminary images to be captured can be reduced one by one.

  When the preliminary imaging adjustment means 22 determines that the preliminary strokes 8a and 8b are too small with respect to the focal position shift amount 20 of the objective lens 4, the preliminary stroke is based on the shortage of the preliminary imaging number. Can be increased.

[Example 3]
Hereinafter, another embodiment of the present invention will be described with reference to FIG. In the following description, the same or equivalent components as those in the other embodiments described above are denoted by the same reference numerals, and the description thereof is simplified or omitted.

  In the first embodiment, a case has been described in which after the slice images for the imaging stroke 9 are all acquired, the image recognition unit 17 detects slice images (front surface image and back surface image) at each of the front surface position and the back surface position of the sample 3. On the other hand, in this embodiment, the slice image formed by the image processing unit 15 is sequentially identified by the image recognition unit 17 and the front image and the back image are detected during imaging at each position in the Z direction of the sample 3. think of.

  The microscope apparatus 1 according to the present embodiment gives an instruction to the stage control unit 14 and the image sensor control unit 13 to stop the imaging operation when the image recognition unit 17 detects the front image or the back image. Stop means 23 is provided. For example, as shown in FIG. 2, when imaging is started from the non-sample imaging range 11 a on the front surface side of the sample 3, the imaging operation is terminated by the imaging stop unit 23 when the back image is detected by the image recognition unit 17. Let Thereby, since the non-sample image on the back surface side of the sample 3 is not acquired, the non-sample image recording means 18 only needs to record the fluctuation amount of the number of non-sample images on the front surface side of the sample 3. The increase in the amount and the imaging time can be suppressed.

[Example 4]
Hereinafter, another embodiment of the present invention will be described with reference to FIG. In the following description, the same or equivalent components as those in the other embodiments described above are denoted by the same reference numerals, and the description thereof is simplified or omitted.

  In the first embodiment, the sample stroke 7 is set by the condition setting unit 2 based on the thickness information of the sample 3 acquired in advance outside. Here, a general sample 3 (biological tissue or the like) is sliced to a thickness of about 5 μm, for example, but in actuality, a variation of several μm occurs with respect to the thickness of each sample 3. Therefore, it is necessary to determine the sample stroke 7 after acquiring the thickness information for each sample 3 to be imaged.

  Therefore, in this embodiment, a thickness measurement area 25 is provided inside the microscope apparatus 1, and the thickness information of the sample 3 is acquired in the thickness measurement area 25. According to this configuration, the thickness information of the sample 3 can be acquired in the thickness measurement area 25 before acquiring the slice image of the sample 3 in the main imaging area 24 including the objective lens 4 and the imaging element 12. At this time, the stage 5 on which the sample 3 is arranged can be controlled not only in the Z direction but also in the XY direction, and reciprocates between the main imaging area 24 and the thickness measurement area 25.

  In the thickness measurement area 25, a stage position measuring device 26 that measures the position of the stage 5 in the Z direction and a sample position measuring device 27 that measures the position of the sample 3 in the Z direction are provided. The stage position measuring device 26 and the sample position measuring device 27 are fixed on the same surface plate so that their relative positions do not shift. The thickness information of the sample 3 is based on the stage height information acquired by the stage position measuring device 26 and the height information of the sample 3 acquired by the sample position measuring device 27 from the upper surface of the stage 5 to the surface of the sample 3. Is calculated as the distance. Then, the sample 3 is moved to the main imaging area 24 by the stage 5, and the sample stroke 7 is set by the condition setting means 2 based on the calculated thickness information of the sample 3. Therefore, according to the microscope apparatus 1 according to the present embodiment, it is possible to obtain thickness information for each of the plurality of samples 3 and set the sample stroke 7 based on each thickness information.

  In this embodiment, in order to acquire thickness information, two measuring instruments, a stage position measuring instrument 26 and a sample position measuring instrument 27, are provided, but the method for acquiring the thickness information of the sample 3 is limited to this. It is not a thing. For example, the height information of the sample 3 and the stage 5 may be acquired by providing only one measuring instrument and moving the stage 5 in the XY direction relative to the measuring instrument.

[Other Examples]
The preferred embodiments of the present invention have been described above, but the present invention is not limited to these embodiments, and various modifications and changes can be made within the scope of the gist.

  For example, in the first embodiment, the sampling pitch for obtaining slice images is provided at equal intervals, but the present invention is not limited to this. That is, it can be arbitrarily set according to the sample 3 to be observed, such as decreasing the sampling pitch of the sample stroke 7 and increasing the sampling pitch of the preliminary strokes 8a and 8b.

  In the first embodiment, the preliminary strokes 8a and 8b are set to be equal to each other. However, the present invention is not limited to this, and the upper and lower sides of the sample stroke 7 may be set to different values. Good. Further, the preliminary stroke may be provided only on either the upper side or the lower side of the sample stroke 7. For example, in the specific example of the first embodiment, the preliminary strokes 8a and 8b are set to 2 μm, respectively, but only one of the preliminary strokes 8a and 8b is provided and the preliminary stroke is set to 4 μm. be able to. Even in such a case, by setting the imaging start position of the stage 5 so that the sample imaging range 10 shown in FIG. The amount of variation can be calculated.

  The correction amount calculation unit 19 according to the first embodiment is configured to correct the imaging range when the number of non-sample images reaches a preset allowable number. However, variations in the number of non-sample images may occur due to noise or the like (rapid temperature fluctuation or the like). For this reason, it is desirable to determine the correction value in advance in consideration of noise or the like and perform the filter processing. For example, the imaging range correction amount 21 is obtained using the result of moving average processing of the fluctuation amount of the number of non-sample images acquired continuously. It is good also as a structure.

  Each of the above-described embodiments may be combined, and the preliminary imaging adjustment unit 22, the imaging stop unit 23, and the thickness measurement area 25 may be arbitrarily provided for the microscope apparatus 1 according to the first embodiment. Can do. Even if the imaging stop unit 23 is not provided as in the third embodiment, the slice image may be sequentially recognized by the image recognition unit 17 during the imaging of the sample 3, and the front image and the back image may be detected. .

DESCRIPTION OF SYMBOLS 1 Microscope apparatus 2 Condition setting means 3 Sample 4 Objective lens 5 Stage 12 Image sensor 17 Image recognition means 18 Non-sample image recording means

Claims (7)

An objective lens that forms an image of the sample, an imaging element that images the sample imaged by the objective lens, and a stage that holds and moves the sample, and
A microscope apparatus for acquiring a plurality of images of the sample by moving the stage in the optical axis direction of the objective lens and imaging the sample at a plurality of positions different in the optical axis direction,
Image recognition means for identifying a sample image including the sample image and a non-sample image not including the sample image of the plurality of images;
A non-sample image recording means for acquiring a variation amount of the number of the non-sample images,
A microscope apparatus, wherein an imaging start position of the stage is adjusted based on the fluctuation amount. Condition setting means is provided for setting a sample stroke, which is the amount of movement of the stage according to the thickness information of the sample, and a preliminary stroke, which is the amount of movement of the stage, according to the preliminary imaging range with respect to the sample. And
The microscope apparatus according to claim 1, wherein the stage is moved in the optical axis direction based on the preliminary stroke and the sample stroke to acquire the plurality of images. Preliminary imaging adjustment means for calculating a preliminary correction value based on the variation amount;
The microscope apparatus according to claim 2, wherein the condition setting unit sets the preliminary stroke based on the preliminary correction value. Having a measuring instrument to obtain thickness information of the sample;
4. The microscope apparatus according to claim 2, wherein the condition setting unit sets the sample stroke based on thickness information of the sample acquired by the measuring instrument.   5. The image capturing unit according to claim 1, further comprising: an imaging stop unit that terminates an imaging operation when the image recognition unit identifies an image at the front surface position or the back surface position of the sample among the plurality of images. The microscope apparatus according to item 1.   The microscope apparatus according to claim 1, wherein the image recognition unit identifies the sample image and the non-sample image based on respective contrasts of the plurality of images. . An objective lens that forms an image of the sample, an imaging element that images the sample imaged by the objective lens, and a stage that holds and moves the sample, and
A microscope apparatus for acquiring a plurality of images of the sample by moving the stage in the optical axis direction of the objective lens and imaging the sample at a plurality of positions different in the optical axis direction,
Image recognition means for identifying a sample image including the sample image and a non-sample image not including the sample image of the plurality of images;
A non-sample image recording means for acquiring a variation amount of the number of the non-sample images,
A microscope apparatus, wherein the necessity of adjustment of the imaging start position of the stage is determined based on the fluctuation amount.

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