JP2018100941A - Method for adjusting regular sample and microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for adjusting a regular sample and a microscope, the method allowing a determination of an insufficient adjustment of an optical element used for a PO observation method before the spindle body in an egg is observed by the PO observation method.SOLUTION: A regular sample SP1 is a regular sample used in a microscope employed for processing the sample by a polarization observation method, and includes a first site SP2 and a second site SP3 next to the first site SP2, the second site having a similar retardation to that of the spindle body in an egg, and the first and second sites SP2 and SP3 having different retardations.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a standard sample used for a microscope for observing a spindle in an egg placed on a stage by a polarization observation method, and a method for adjusting a microscope using the standard sample.

  Conventionally, microinsemination in the field of advanced reproductive medicine is known as an application of a microscope. Microinsemination is a method of insemination of sperm and ovum under a microscope. For example, a sperm injection method (intracytoplasmic sperm injection) in which a micropipette containing sperm is inserted into an egg fixed with a holding pipette and the sperm is injected into the egg ( Intracytoplasmic Sperm Injection (hereinafter referred to as “ICSI”) is known. In this ICSI, in order to operate a specimen on the stage, it is common to use an inverted microscope having a large working space above the stage.

  In recent years, in the field of micro insemination, in order to improve the insemination rate of eggs, a method of micro insemination using a microscope while appropriately switching a plurality of observation methods has attracted attention. For example, a relief contrast observation method (hereinafter referred to as “RC observation method”) capable of observing an ovum in three dimensions and a polarization observation method (hereinafter referred to as “PO observation method”) capable of observing the position of the spindle of the ovum. In addition, a method of using while switching according to the observation purpose is becoming widespread (for example, see Patent Document 1).

JP 2014-92641 A

  By the way, in the conventional microscope mentioned above, when a user observes by PO observation method, the spindle in an ovum may not be confirmed. The main causes are those caused by the state of the ovum and those caused by insufficient adjustment of the optical elements of the microscope used in the PO observation method. Among these causes, if the adjustment state of the optical element of the microscope used in the PO observation method can be confirmed, if the spindle in the ovum cannot be confirmed, it can be determined that it is caused by the state of the ovum. In addition, the spindle in the ovum sometimes exists at a position different from the standard position. In such a case, it is difficult to observe a plurality of ova continuously in the same adjustment state. is there. For this reason, the technique which can confirm the adjustment state of the optical element of the microscope used by PO observation method under microscope observation was desired.

  The present invention has been made in view of the above, and a standard sample and a microscope capable of determining the lack of adjustment of optical elements used in the PO observation method before confirming the spindle in the ovum by the PO observation method It aims at providing the adjustment method of.

  In order to solve the above-described problems and achieve the object, a standard sample according to the present invention is a standard sample used for adjustment of a microscope that performs treatment on a sample by polarized light observation, and includes the first part, A second portion adjacent to the first portion and having a retardation equivalent to that of the spindle in the ovum, wherein the first portion and the second portion are different in retardation.

  The standard sample according to the present invention is characterized in that, in the above invention, the standard sample has an outer diameter of 10 mm or less.

  The standard sample according to the present invention is characterized in that, in the above invention, the standard sample has an outer diameter of 5 ± 2 mm or less.

  The standard sample according to the present invention is characterized in that, in the above invention, the retardation range of the second part is 2 to 5 nm.

  In the standard sample according to the present invention, in the above invention, the first part has retardation equivalent to that of a living tissue.

  Further, the standard sample according to the present invention is characterized in that, in the above invention, the standard sample has a part or all of an annular appearance.

  The standard sample according to the present invention is characterized in that, in the above invention, the first part and the second part are stained.

  The standard sample according to the present invention is characterized in that, in the above invention, the first part and the second part are fixed by a tissue fixing solution.

  The standard sample according to the present invention is characterized in that, in the above invention, the first part and the second part are derived from at least a testis.

  The standard sample according to the present invention is characterized in that, in the above invention, the second part is a thin film region covering an outer periphery of the testis.

  A microscope adjustment method according to the present invention is a microscope adjustment method including a plurality of optical elements used in a polarization observation method. And adjusting the contrast of the observation image by operating any one or more of the plurality of optical elements when the change in the contrast of the observation image of the two parts cannot be observed.

  According to the present invention, there is an effect that it is possible to determine whether the optical element used in the PO observation method is insufficiently adjusted before the spindle in the ovum is confirmed by the PO observation method.

FIG. 1 is a diagram showing a tissue section of a standard sample according to an embodiment of the present invention. FIG. 2A is an enlarged view enlarging the region A of FIG. 1 before the polarization contrast is inverted. FIG. 2B is an enlarged view enlarging region A of FIG. 1 after the polarization contrast is inverted. FIG. 3 is a flowchart showing an outline of a method for manufacturing a standard sample according to an embodiment of the present invention. FIG. 4A is an enlarged view before inversion of the polarization contrast with respect to the standard sample SP1 fixed with the Bouin liquid. FIG. 4B is an enlarged view after inversion of the polarization contrast fixed with the Bouin liquid. FIG. 5A is an enlarged view before inversion of polarization contrast with respect to the standard sample SP1 fixed by a general tissue fixing method. FIG. 5B is an enlarged view after the inversion of the polarization contrast with respect to the standard sample SP1 fixed by a general tissue fixing method. FIG. 6A is an enlarged view of the standard sample SP1 subjected to hematoxylin staining before inversion of polarization contrast. FIG. 6B is an enlarged view after reversal of the polarization contrast with respect to the standard sample SP1 subjected to hematoxylin staining. FIG. 7A is an enlarged view of the standard sample SP1 subjected to hematoxylin and eosin staining before inversion of polarization contrast. FIG. 7B is an enlarged view after inversion of polarization contrast with respect to the standard sample SP1 subjected to hematoxylin and eosin staining. FIG. 8A is an enlarged view of the standard sample SP1 subjected to hematoxylin and eosin staining before inversion of polarization contrast. FIG. 8B is an enlarged view after inversion of polarization contrast with respect to the standard sample SP1 subjected to hematoxylin and eosin staining. FIG. 9 is a conceptual diagram showing a configuration of a microscope according to an embodiment of the present invention. FIG. 10 is a block diagram showing a schematic configuration of a microscope according to an embodiment of the present invention. FIG. 11 is a diagram showing the configuration of the condenser turret of the microscope shown in FIG. FIG. 12 is a plan view of a petri dish including a specimen. 13 is a cross-sectional view taken along line AA in FIG. FIG. 14 is a diagram schematically showing the configuration of the operation input unit of the microscope according to the embodiment of the present invention. FIG. 15 is a diagram showing setting information recorded by the setting information recording unit in the recording unit of the microscope shown in FIG. FIG. 16 is a diagram schematically showing the arrangement of units in the PO observation method performed by the microscope according to the embodiment of the present invention. FIG. 17 is a flowchart showing an outline of a microscope adjustment method according to an embodiment of the present invention.

  Hereinafter, a standard sample according to an embodiment of the present invention will be described with reference to the drawings. In the following description, a microscope capable of ICSI is exemplified, but the present invention is not limited to this embodiment. Moreover, in the following description, each figure has shown only the shape, magnitude | size, and positional relationship roughly to such an extent that the content of this invention can be understood. Therefore, the present invention is not limited only to the shape, size, and positional relationship exemplified in the drawings. In the description of the drawings, the same portions are denoted by the same reference numerals.

[Standard sample]
FIG. 1 is a diagram showing a tissue section of a standard sample according to an embodiment of the present invention. FIG. 2A is an enlarged view enlarging the region A of FIG. 1 before the polarization contrast is inverted. FIG. 2B is an enlarged view enlarging region A of FIG. 1 after the polarization contrast is inverted. FIG. 1 shows a situation where PO observation is performed at an observation magnification of 20 times.

  The standard sample SP1 shown in FIG. 1, FIG. 2A and FIG. 2B includes a first part SP2 and a second part SP3 adjacent to the first part SP2 and having a retardation equivalent to that of the spindle in the ovum. Prepare. The first part SP2 has retardation (a phase difference optically generated by birefringence) equivalent to that of a living tissue. Here, the retardation equivalent to the biological tissue is assumed to be the cell region of the ovum that is the background of the spindle, and is less than 2 nm, and in some cases zero (when the phase difference is substantially zero). obtain. Moreover, the retardation equivalent to the spindle in an ovum has a retardation range of 2 to 5 nm. That is, the first part SP2 and the second part SP3 have different retardations. Further, the standard sample SP1 has an annular appearance as an observation image, preferably a circle or an ellipse, and its outer diameter d may be 10 mm or less, but is preferably 5 ± 2 mm or less. Note that the shape of the standard sample SP1 does not have to be annular. For example, a part of the annular portion of the standard sample SP1 may be cut out and used. By using a part or the whole of the annular shape, different retardations are adjacent to each other as in the case of an ovum, so that there is an advantage that adjustment work by microscopic operation can be easily performed. The standard sample SP1 is derived from the mouse testis. Here, testicular origin is derived from mammals such as mice, pigs, cows and horses, and humans are excluded. Of course, as testicular origin, for example, amphibians and fish may be used. Further, the first part SP2 is a testicular septum, and the second part SP3 is a thin film region (white film) covering the outer periphery of the testicular septum.

  The standard sample SP1 is fixed with a tissue fixing solution. The tissue fixative is preferably bouin's fluid. Furthermore, the first part SP2 and the second part SP3 are stained with a staining solution. As the staining solution, a hematoxylin solution (Hematoxylin) and a hematoxylin-eosin solution (Hematoxylin-Eosin) are preferably used. More preferably, a hematoxylin solution is used. In addition, the standard sample SP1 is placed on the slide glass SP4, filled with an encapsulant, and covered with a cover glass (not shown).

[Manufacturing method of standard sample]
Next, a method for manufacturing the standard sample SP1 will be described. FIG. 3 is a flowchart showing an outline of a manufacturing method of the standard sample SP1. In the following, in order to explain the differences in the effects of each of the first part SP2 and the second part SP3 in the standard sample SP1 and the effects of each reagent, PO observation is performed on the already prepared standard sample SP1. An explanation will be given by using supplementary enlarged views before and after reversing the polarization contrast of the standard sample SP1 when observed by the method.

  As shown in FIG. 3, first, the user obtains a mouse testis (step S1), and fixes and embraces it with a bouin solution (step S2).

  FIG. 4A is an enlarged view before inversion of the polarization contrast with respect to the standard sample SP1 fixed with the Bouin liquid. FIG. 4B is an enlarged view after inversion of the polarization contrast fixed with the Bouin liquid. FIG. 5A is an enlarged view before inversion of polarization contrast with respect to the standard sample SP1 fixed by a general tissue fixing method. FIG. 5B is an enlarged view after the inversion of the polarization contrast with respect to the standard sample SP1 fixed by a general tissue fixing method.

  As shown in FIG. 4A and FIG. 4B, when the mouse testis is fixed with Bouin's solution, the second part SP3 is not detached from the tissue of the first part SP2 in the central part composed of the testis canal and the like. . On the other hand, as shown in FIG. 5A and FIG. 5B, when the formalin solution or paraformaldehyde solution used in a general tissue fixing method is used, the testis of the mouse is changed from the first part SP2 to the second part. SP3 peels off. In this case, in the second portion SP3, the portion where the polarization image is observed black and the portion observed white are mixed in the same field of view of the microscope. For this reason, when a user observes the testis of a mouse fixed by a general tissue fixing method with a microscope using the PO observation method, the user observes the spindle in white when observing the spindle in the ovum using the PO observation method. It cannot be judged whether it should be observed or observed black. However, as shown in FIG. 4A and FIG. 4B, when the user observes the spindle in the ovum after observing the standard sample SP1 with the microscope by the PO observation method, the first and the second before and after contrast inversion. Since the contrast of the part SP2 and the second part SP3 is inverted separately, the first part SP2 and the second part SP3 can be easily identified.

Returning to FIG. 3, the description after step S2 will be continued.
After step S2, the user prepares a tissue section using the testis of a mouse fixed and encapsulated with Buan's solution (step S3).

  Subsequently, hematoxylin staining is performed on a tissue section placed on a slide glass or petri dish using a hematoxylin solution (step S4).

  FIG. 6A is an enlarged view of the standard sample SP1 subjected to hematoxylin staining before inversion of polarization contrast. FIG. 6B is an enlarged view after reversal of the polarization contrast with respect to the standard sample SP1 subjected to hematoxylin staining. FIG. 7A is an enlarged view of the standard sample SP1 subjected to hematoxylin and eosin staining before inversion of polarization contrast. FIG. 7B is an enlarged view after inversion of polarization contrast with respect to the standard sample SP1 subjected to hematoxylin and eosin staining. FIG. 8A is an enlarged view of the standard sample SP1 subjected to hematoxylin and eosin staining before inversion of polarization contrast. FIG. 8B is an enlarged view after inversion of polarization contrast with respect to the standard sample SP1 subjected to hematoxylin and eosin staining.

  As shown in FIG. 6A and FIG. 6B, when a tissue section is stained with hematoxylin using a hematoxylin solution, a desired polarized image can be observed with respect to the second part SP3 (white film). On the other hand, as shown in FIGS. 7A and 7B, when a tissue section is stained with hematoxylin and eosin using a general hematoxylin and eosin solution, a predetermined polarization image (white polarization image) is displayed on the second region SP3. ) And a different polarization image (yellow polarization image) are observed. This yellow polarized image is significantly different from the white observed image (spindle) observed on the spindle in the ovum. The white membrane of the mouse testis is eosin positive and may have some polarization characteristics by eosin staining, but even when hematoxylin and eosin solution is used as shown in FIGS. 8A and 8B. In some cases, a desired polarized image can be observed with respect to the second part SP3 (white film). Therefore, in one embodiment of the present invention, the tissue section is preferably stained with a hematoxylin-eosin solution or a hematoxylin solution, and more preferably with a hematoxylin solution. desirable.

Returning to FIG. 3, the description of step S4 and subsequent steps will be continued.
After step S4, the user places the cover glass by pouring the mounting medium on the tissue section on the slide glass that has been stained with hematoxylin (step S5).

  Thereafter, the user performs PO observation on the standard sample SP1 using a microscope capable of PO observation (step S6). In this case, the user visually determines whether or not the standard sample SP1 has been properly manufactured by observing retardations of the first part SP2 and the second part SP3 that are different from each other. After step S6, the production of the standard sample SP1 is finished.

  An optical element used in the PO observation method is obtained by observing the second portion SP3 by the PO observation method before confirming the spindle in the egg by the PO observation method. The lack of adjustment can be determined.

  Next, a method for adjusting the microscope when performing the ICSI with the microscope using the standard sample SP1 described above will be described. In the following, prior to the description of the method for adjusting the microscope, the configuration of the microscope according to the embodiment of the present invention will be described, and then the method for adjusting the microscope according to the embodiment of the present invention will be described.

[Configuration of microscope]
FIG. 9 is a conceptual diagram showing a configuration of a microscope according to an embodiment of the present invention. FIG. 10 is a block diagram showing a schematic configuration of a microscope according to an embodiment of the present invention. 9 and 10, the plane on which the microscope 1 is placed will be described as an XY plane, and a direction perpendicular to the XY plane will be described as a Z direction.

  9 and 10, the microscope main body 2 that observes the petri dish 100 in which the specimen SP is accommodated, the operation input unit 3 that receives input of various operations of the microscope 1, and the microscope main body 2 capture images. A display unit 4 for displaying an image corresponding to the image data, a recording unit 5 for recording various programs and parameters for driving the microscope 1, and a control unit 6 for controlling the microscope main body unit 2 and the display unit 4. Prepare. The microscope main body 2, the operation input unit 3, the display unit 4, the recording unit 5, and the control unit 6 are connected by wire or wireless so that data can be transmitted and received.

  First, the microscope main body 2 will be described in detail. The microscope main body 2 includes a light source 10, a polarizer 11, a compensator 12, a condenser turret 13, a condenser lens 14, a stage 15, a stage position detector 16, a revolver 17, an objective lens 18, and a revolver position. A detection unit 19, a DIC prism 20, an analyzer 21, an imaging lens 22, an optical path splitting prism 23, an imaging unit 24, a mirror 25, an eyepiece lens 26, and a drive unit 27 are provided.

  The light source 10 includes a halogen lamp, a xenon lamp, an LED (Light Emitting Diode), or the like. The light source 10 emits illumination light toward the specimen SP.

  The polarizer 11 is disposed on the optical axis XA between the light source 10 and the compensator 12 and transmits only the polarization component in one direction of the illumination light emitted by the light source 10. The polarizer 11 is disposed so as to be rotatable about the optical axis XA of the optical path of the light source 10. The polarizer 11 is configured using a polarizing plate that is one of optical elements such as a filter. Further, the polarizer 11 is rotated around the optical axis XA by a motor 11a configured by a stepping motor, a DC motor, or the like under the drive control of the drive unit 27.

  The compensator 12 is an optical element for measuring the phase difference (retardation) due to the anisotropy of the specimen SP, and adjusts the retardation of the light transmitted through the polarizer 11. The compensator 12 is disposed on the optical path between the condenser lens 14 and the polarizer 11 so as to be rotatable about the optical axis of the objective lens 18. The compensator 12 is configured using a liquid crystal or a wave plate. Specifically, the compensator 12 is configured using a bereak compensator, a senal mon type compensator, a brace scaler compensator, a quartz wedge compensator, and a liquid crystal modulation element. The compensator 12 is preferably a liquid crystal modulation element, a senalmon type compensator, or a brace scaler compensator because it is desirable that the visual field retardation is substantially uniform when performing the PO observation method for observing the ovum spindle. When a liquid crystal modulation element is used as the compensator 12, the retardation can be changed by electrically controlling the liquid crystal molecules. When a Senalmon type compensator is used as the compensator 12, the retardation of the compensator 12 can be changed by the rotation of the polarizer 11 with respect to the wave plate in the compensator 12. Further, when a brace scaler compensator is used as the compensator 12, the retardation of the compensator 12 can be changed by the rotation of the prism in the compensator 12. Furthermore, the compensator 12 is rotated around the optical axis XA by a motor 12a configured by a stepping motor, a DC motor, or the like under the drive control of the drive unit 27.

  The condenser turret 13 has a plurality of optical elements that are used by switching according to the observation method and the magnification, and is arranged rotatably on the optical axis XA. The condenser turret 13 is rotated in accordance with the observation method, so that one of the optical elements is arranged on the optical axis XA. The capacitor turret 13 is rotated by a motor 13 a configured by a stepping motor, a DC motor, or the like under the drive control of the drive unit 27.

  FIG. 11 is a diagram illustrating a configuration of the capacitor turret 13. As shown in FIG. 11, the capacitor turret 13 includes an opening 130, an RC observation aperture plate 131, an RC observation aperture plate 132, and a DIC prism 133.

  The opening 130 together with the capacitor turret 13 constitutes an opening plate (hole). The opening 130 is formed with a sufficient size that does not block the illumination light from the light source 10, and realizes illumination with a high numerical aperture. The opening 130 is used, for example, when the microscope 1 performs bright field observation or PO observation method. Specifically, when the microscope 1 performs bright field observation, the opening 130 is operated by a user searching for a place in the petri dish 100 or using a manipulator using a 4 × or 10 × objective lens 18 in preparation for microinsemination. This is used when positioning the tip of a micropipette to be performed.

  The RC observation aperture plate 131 is an aperture plate used for RC observation, and has a polarizing plate 131b in a part of the aperture 131a formed at a position eccentric from the optical axis XA when arranged on the optical axis XA. . The opening 131a is formed at a position shifted from the center of the RC observation opening plate 131, thereby realizing oblique illumination. The RC observation aperture plate 131 is used, for example, when the microscope 1 performs a 20-fold RC observation method.

  The RC observation aperture plate 132 is an aperture plate used for RC observation, and has a polarizing plate 132b in a part of the aperture 132a formed at a position eccentric from the optical axis XA when arranged on the optical axis XA. . The opening 132a is formed at a position shifted from the center of the RC observation opening plate 132, thereby realizing oblique illumination. The RC observation aperture plate 132 is used, for example, when the microscope 1 performs a 40-fold RC observation method.

  The DIC prism 133 is arranged on the optical axis XA by the rotation of the condenser turret 13. The DIC prism 133 is paired with a DIC prism 20 disposed on the image side on the objective lens 18 side described later, and constitutes a differential interference optical system. The DIC prism 133 is configured using a Nomarski prism or the like. The DIC prism 133 is used, for example, when the microscope 1 performs a 60-fold DIC observation method.

  In the thus configured condenser turret 13, the optical element arranged on the optical axis XA is switched by rotating the condenser turret 13 by the motor 13a according to the observation method. Specifically, when performing RC observation, the condenser turret 13 has the RC observation aperture plate 131 or the RC observation aperture plate 132 disposed on the optical axis XA. When performing the DIC observation method, the DIC prism 133 is optically coupled. When the bright field observation method or the PO observation method is performed on the axis XA, the aperture 130 is disposed on the optical axis XA.

  The condenser lens 14 is disposed on the optical axis XA, collects the illumination light emitted from the light source 10, and uniformly irradiates the region including the sample SP in the petri dish 100. The condenser lens 14 may include a field stop that can adjust the amount of illumination light emitted from the light source 10 and a field stop operation unit that changes the diameter of the field stop.

  The stage 15 is configured to be movable in the XYZ directions. The stage 15 is moved in the XY plane and in the Z direction by the drive unit 27. On the stage 15, the petri dish 100 on which the specimen S is placed is placed. Under the control of the control unit 6, the stage 15 detects a predetermined origin position on the XY plane by the stage position detection unit 16, and the driving amount of the drive unit 27 is limited based on this origin position, thereby allowing the sample Move to a desired observation location (observation region) on the SP. Further, the stage 15 is detected by the stage position detection unit 16 in the Z direction under the control of the control unit 6, and the drive amount of the drive unit 27 is limited based on this position. The condenser lens 14 and the objective lens 18 are moved to a focus position (focus position). The stage 15 is moved in the XY plane and in the Z direction by a motor 15a configured by a stepping motor, a DC motor, or the like under the drive control of the drive unit 27. The stage 15 may be provided with a heating unit that holds the petri dish 100 at a constant temperature. Moreover, the stage 15 does not need to be electrically driven and may be manually movable.

  Here, the petri dish 100 in which the specimen S is arranged will be described in detail. FIG. 12 is a plan view of the petri dish 100 including the specimen S. FIG. 13 is a cross-sectional view taken along line AA in FIG. As shown in FIGS. 12 and 13, the petri dish 100 used for microinsemination is formed with an ICSI drop R1 (culture solution) for inseminating sperm into an egg and a sperm selection drop R2 (culture solution) for selecting sperm. Each drop is covered with mineral oil Wa which prevents air from being infected with bacteria and drying. The number of drops on the petri dish 100 can be changed as appropriate.

  The stage position detection unit 16 is configured using an encoder, an optical photo interrupter, or the like, detects the stage position of the stage 15 in the XY plane and the Z direction of the stage 15, and outputs the detection result to the control unit 6. The stage position detection unit 16 detects the stage position of the stage 15 based on the number of pulses of the drive unit 27 that is driven according to the drive signal input from the control unit 6, and sends the detection result to the control unit 6. It may be output.

  The revolver 17 is equipped with a plurality of objective lenses 18. The revolver 17 is provided so as to be rotatable with respect to the optical axis XA, and the objective lens 18 is disposed below the sample SP. The revolver 17 is configured using a swing revolver or the like. The revolver 17 is rotated by a motor 17 a configured by a stepping motor, a DC motor, or the like under the drive control of the drive unit 27. The revolver 17 is provided so as to be movable along the optical axis XA direction, and is moved up and down in the Z direction by the drive unit 27. Note that a focusing mechanism that moves the sample side in the direction of the optical axis XA may be provided separately from the revolver 17.

  The objective lens 18 is disposed at a position on the optical axis XA facing the condenser lens 14 with the sample S interposed therebetween. The objective lens 18 includes an objective lens 181, an objective lens 182, and an objective lens 183. The objective lens 181 is an objective lens having a magnification suitable for observing an egg, for example, a low magnification such as 10 times or 20 times, and is used for the RC observation method. The objective lens 181 has a modulator 1811 having three regions with different transmittances at the pupil position of the objective lens 181. The modulator 1811 includes a region 1811a having a transmittance of 100%, a region 1811b having a transmittance of approximately 25%, and a region 1811c having a transmittance of 0%. The modulator 1811 has an optically conjugate relationship with the RC observation aperture plate 131 and the RC observation aperture plate 132 disposed at the pupil position of the condenser lens 14. The objective lens 181 is also applied to a PO observation method in which an ovum spindle is a main observation target and approximately the same magnification is required. The objective lens 182 is an objective lens having a magnification suitable for sperm observation, for example, a high magnification such as 60 times or 100 times, and is used in the DIC observation method. The objective lens 183 is an objective lens having a magnification suitable for observing the tip of a micropipette, for example, a low magnification of 4 times, and is used for bright field observation.

  The revolver position detector 19 detects the position of the revolver 17 in the Z direction and outputs the detection result to the controller 6. The revolver position detector 19 is configured using an encoder, an optical photo interrupter, or the like. The revolver position detection unit 19 detects the position of the revolver 17 in the Z direction based on the number of pulses of the drive unit 27 driven in accordance with the drive signal input from the control unit 6, and the detection result is used as a control unit. 6 may be output.

  The DIC prism 20 is paired with the DIC prism 133 and constitutes a differential interference optical system. The DIC prism 20 is configured using a Nomarski prism or the like. The DIC prism 20 is detachably disposed on the optical axis XA between the objective lens 18 and the analyzer 21. Further, the DIC prism 20 is arranged on the optical axis XA by a motor 20 a configured by a stepping motor, a DC motor, or the like under the drive control of the drive unit 27.

  The analyzer 21 is disposed on the observation-side optical axis XA at the rear stage of the objective lens 18 and transmits only the polarization component in one direction of the light transmitted through the sample SP according to the relative positional relationship with the polarizer 11. Specifically, the polarizer 11 and the analyzer 21 are arranged so as to be in a crossed Nicols state in which the polarization directions are orthogonal to each other. Further, the analyzer 21 is arranged so as to be in a 45-degree direction with respect to the vibration direction of the polarizing plate 131 b of the RC observation aperture plate 131 of the capacitor turret 13. Thereby, the microscope 1 can perform the RC observation method without any trouble in the observation. The analyzer 21 may be arranged so as to be able to advance and retreat on the optical axis XA.

  The imaging lens 22 condenses the light emitted from the objective lens 18 and forms an observation image. The imaging lens 22 is configured using one or a plurality of lenses.

  The optical path dividing prism 23 divides the light of the observation image formed by the imaging lens 22 into the imaging unit 24 and the mirror 25. The optical path splitting prism 23 is configured by using a prism having a coating for splitting light on the joint surface.

  The imaging unit 24 is configured using an imaging element such as a CCD (Charge Coupled Device) or a CMOS (Complementary Metal Oxide Semiconductor), and captures an observation image of the specimen SP that has entered through the imaging lens 22 and the optical path splitting prism 23. Then, image data is generated, and this image data is output to the control unit 6.

  The mirror 25 reflects the observation image emitted from the imaging lens 22 toward the eyepiece lens 26. A plurality of relay lenses may be provided on the optical path between the mirror 25 and the eyepiece lens 26.

  The eyepiece 26 enlarges the observation image incident through the imaging lens 22, the optical path splitting prism 23, and the mirror 25. The eyepiece 26 is configured using one or a plurality of lenses.

  The drive unit 27 is configured using a drive driver, and moves or rotates each optical element of the microscope body 2 under the control of the control unit 6. Specifically, the drive unit 27 drives the motor 11a, the motor 12a, the motor 13a, the motor 15a, the motor 17a, and the motor 20a under the control of the control unit 6, thereby causing the polarizer 11, the compensator 12, and the capacitor to be driven. The turret 13, the stage 15, the revolver 17, the DIC prism 20, and the analyzer 21 are rotated or moved to predetermined positions.

  The operation input unit 3 receives input of various operations of the microscope 1. The operation input unit 3 is configured using a keyboard, a mouse, a joystick, a touch panel, various buttons, and the like, and outputs an operation signal corresponding to operation inputs of various switches to the control unit 6.

  FIG. 14 is a diagram illustrating a configuration of the operation input unit 3. As shown in FIG. 14, the operation input unit 3 includes buttons B <b> 1 to B <b> 5 that receive input of instruction signals for instructing each observation method, buttons B <b> 6 and B <b> 7 that receive input of instruction signals for adjusting contrast, and magnification of the objective lens 18. Buttons B8 to B13 for receiving an input of an instruction signal for instructing, a button B14 for receiving an input of an instruction signal for registering the position of the stage 15, a button B15 for receiving an input of an instruction signal for instructing movement of the XY plane of the stage 15, and There is a button B16 for receiving an input of an instruction signal instructing the movement of the stage 15 in the Z direction.

  The display part 4 is comprised using the display panel which consists of a liquid crystal or organic EL (Electro Luminescence). The display unit 4 displays an image corresponding to the image data input from the imaging unit 24 via the control unit 6.

  The recording unit 5 records various programs to be executed by the microscope 1 and various data used during the execution of the programs. The recording unit 5 is configured using a semiconductor memory such as a flash memory and a RAM (Random Access Memory). Further, the recording unit 5 includes arrangement information of each optical element on the optical axis XA for each observation.

  FIG. 15 is a diagram illustrating setting information recorded by the setting information recording unit 51. As shown in FIG. 15, in the setting information T1, position information of each optical element corresponding to each observation method is recorded. Specifically, as shown in FIG. 15, when performing the bright field observation method, the polarizer 11 is arranged in a paranicol state with respect to the analyzer 21, and the compensator 12 is placed on the optical axis XA with the condenser turret 13. A hole (opening 130) is disposed on the optical axis XA, the objective lens 18 is quadrupled, the DIC prism 20 is disposed outside the optical axis XA, and the analyzer 21 is disposed on the optical axis XA.

  The control unit 6 is configured using a CPU (Central Processing Unit) or the like, and comprehensively controls the operation of each unit configuring the microscope 1. The control unit 6 drives each unit constituting the microscope 1 according to the operation signal input from the operation input unit 3. Specifically, when an instruction signal for instructing bright field observation is input from the operation input unit 3, the control unit 6 refers to the setting information recorded by the setting information recording unit 51 and determines the polarizer according to the observation method. 11, the compensator 12, the condenser turret 13, the stage 15, the revolver 17, the DIC prism 20, and the analyzer 21 are driven and arranged on the optical axis XA to change to an instruction signal observation method. For example, when the DIC observation is instructed according to the instruction signal input from the operation input unit 3, the control unit 6 drives the drive unit 27 to rotate the revolver 17 so that the objective lens 182 moves the optical axis XA. The DIC prism 133 and the DIC prism 20 are arranged on the optical axis XA by rotating the condenser turret 13. Further, when the microscope 1 performs the PO observation method, the control unit 6 causes the drive unit 27 to drive the compensator 12 within a range including the position where the retardation becomes 0 as a reference, thereby increasing or decreasing the retardation. Specifically, the control unit 6 causes the driving unit 27 to rotate the compensator 12 repeatedly within an angle range across the position where the retardation becomes zero.

  The microscope 1 configured as described above switches the arrangement positions on the optical axis XA of the polarizer 11, the compensator 12, the condenser turret 13, the revolver 17, the DIC prism 20, and the analyzer 21 under the control of the control unit 6. Thus, a bright field observation method, an RC observation method, and a DIC observation method can be performed. For example, when the microscope 1 performs the observation method by RC observation, the control unit 6 rotates the revolver 17 so as to place the objective lens 181 on the optical axis XA and rotate the condenser turret 13 to obtain the light. An RC observation aperture plate 131 is disposed on the axis XA. Furthermore, the control unit 6 arranges the DIC prism 20 outside the optical axis XA. Thereby, the observation method of the microscope 1 can be switched to the RC observation method. In the case of the RC observation method, the DIC prism 20 may be disposed on the optical axis XA.

  In addition, when the microscope 1 performs the observation method using the DIC observation method, the control unit 6 rotates the revolver 17 to place the objective lens 182 on the optical axis XA and rotate the condenser turret 13. A DIC prism 133 is disposed on the optical axis XA. Further, the control unit 6 drives the drive unit 27 to place the DIC prism 20 on the optical axis XA, and rotates the polarizer 11 around the optical axis XA, thereby causing the polarizer 11 and the analyzer 21 to move. Set to the crossed Nicols state. Thereby, the observation method of the microscope 1 can be switched to the DIC observation method.

  In addition, when the microscope 1 performs the observation method by the PO observation method, the control unit 6 rotates the revolver 17 to place the objective lens 181 on the optical axis XA and rotate the condenser turret 13. An opening 130 (hole) is arranged on the optical axis XA. Further, the control unit 6 rotates the polarizer 11 about the optical axis XA as a rotation axis, thereby bringing the polarizer 11 and the analyzer 21 into a crossed Nicols state (see FIG. 16). Thereby, the control part 6 can switch the observation method of the microscope 1 to PO observation method (20 times). In the case of the PO observation method, the compensator 12 is rotated at an arbitrary angle around the optical axis XA passing through the specimen SP.

[How to adjust the microscope]
Next, an adjustment method when performing the PO observation method of the microscope 1 using the standard sample SP1 described above will be described. FIG. 17 is a flowchart showing an outline of an adjustment method when performing the PO observation method of the microscope 1 using the standard sample SP1 according to the embodiment of the present invention.

  As shown in FIG. 17, first, the user sets an element for PO observation in the microscope 1 by pressing the button B4 of the operation input unit 3 (step S101). Specifically, the control unit 6 sets each element constituting the microscope 1 in a state in which PO observation is possible in accordance with an instruction signal input from the operation input unit 3 (see FIG. 16 described above).

  Subsequently, the user places the standard sample SP1 on the stage 15 and performs PO observation of the standard sample SP1 (step S102).

  Thereafter, the user determines whether or not the target polarized image can be observed with respect to the standard sample SP1 (step S103). Specifically, it is determined whether or not the change in the contrast of the polarization image of the second part SP3 (white film) can be observed with respect to the standard sample SP1. If the target polarized image can be observed with respect to the standard sample SP1 (step S103: Yes), the user ends the adjustment method of the microscope 1 and performs ICSI while observing the ovum spindle. In this case, when the user cannot observe not only the position of the standard spindle in the ovum (near the first polar body of the ovum) but also the whole cell under the microscope, Since it has been adjusted, it is determined that the condition of the ovum is not good, and sperm is injected using another ovum without injecting sperm. On the other hand, when the observed ovum is determined to be in the M1 phase based on the shape or the like, it is desirable to observe the spindle again after further culturing in the incubator. On the other hand, when the target polarized image cannot be observed with respect to the standard sample SP1 (step S103: No), the user returns to step S101 described above. In this case, the user finely adjusts each element used in the PO observation method, for example, the polarizer 11 and the analyzer 21, and again observes the standard sample SP1 to obtain a target polarized image on the standard sample SP1. The elements constituting the microscope 1 are adjusted until they can be observed.

  Thus, before observing the spindle of an ovum, each element constituting the microscope 1 is adjusted while observing the microscope using the standard sample SP1 having a retardation equivalent to that of the spindle, so the adjustment of the microscope 1 is insufficient. Or whether the egg is in poor condition.

  According to the embodiment of the present invention described above, the standard sample SP1 is used to adjust each element constituting the microscope 1 under the microscope observation. Therefore, before the spindle in the ovum is confirmed by the PO observation method. In addition, it is possible to determine the lack of adjustment of the optical element used in the PO observation method. As a result, even if the spindle body is not present at the standard position in the ovum, the position of the spindle body can be probed under a microscope. Moreover, when the spindle in an ovum cannot be confirmed, it can be judged that it originates in the state of an ovum.

  In one embodiment of the present invention, the eggs were observed and treated after adjusting each element of the microscope 1 using the standard sample SP1, but the standard sample is within the same field of view of the microscope 1, for example. The SP1 and the ovum may be placed, and the ovum may be observed and treated while adjusting each element of the microscope 1 within the field of view. Further, regarding the adjustment of each element using the standard sample SP1 and / or the confirmation of the spindle in the ovum, the observation state under the microscope may be directly observed via the eyepiece 26, or the display unit 4 You may observe by the image displayed on. Regarding the adjustment of each element and / or the confirmation of the spindle, the determination result may be automatically output to the display unit 4 by image recognition processing.

  In one embodiment of the present invention, as the standard sample SP1, the state in which the appearance is a circle or an ellipse is reversed from the sign of the curvature as in the case of most natural biological samples. The shape may be a circle or an ellipse that has a shape that is distorted to such an extent that it does not. Further, the standard sample SP1 may be a non-natural sample that is artificially manufactured using a biological material or artificially regenerated from the same biological material by a regeneration technique using stem cells.

DESCRIPTION OF SYMBOLS 1 Microscope 2 Microscope main-body part 3 Operation input part 4 Display part 5 Recording part 6 Control part 10 Light source 11 Polarizer 12 Compensator 13 Condenser turret 14 Condenser lens 15 Stage 16 Stage position detection part 17 Revolver 18 Objective lens 19 Revolver position detection part 20 DIC Prism 21 Analyzer 22 Imaging lens 23 Optical path splitting prism 24 Imaging unit 25 Mirror 26 Eyepiece 27 Drive unit 51 Setting information recording unit 100 Petri dish SP specimen SP1 Standard sample SP2 First part SP3 Second part

Claims (11)

A standard sample used for adjustment of a microscope for treating a sample by polarized light observation method,
A first site;
A second site adjacent to the first site and having a retardation equivalent to the spindle in the ovum;
With
A standard sample, wherein the first part and the second part are different in retardation.   The standard sample according to claim 1, wherein an outer diameter of the standard sample is 10 mm or less.   The standard sample according to claim 2, wherein an outer diameter of the standard sample is 5 ± 2 mm or less.   The standard sample according to any one of claims 1 to 3, wherein a retardation range of the second part is 2 to 5 nm.   The standard sample according to claim 4, wherein the first part has retardation equivalent to that of a living tissue.   The standard sample according to any one of claims 1 to 5, wherein the external appearance of the standard sample is a part or all of an annular shape.   The standard sample according to any one of claims 1 to 6, wherein the first part and the second part are stained.   The standard sample according to claim 7, wherein the first part and the second part are fixed by a tissue fixing solution.   The standard sample according to any one of claims 1 to 8, wherein the first part and the second part are derived from at least a testis.   The standard sample according to claim 9, wherein the second portion is a thin film region covering an outer periphery of the testis. A method for adjusting a microscope provided with a plurality of optical elements used in polarization observation,
When the change in the contrast of the observation image of the second part cannot be observed before and after inversion of the polarization contrast with respect to the standard sample according to any one of claims 1 to 10, any one of the plurality of optical elements A method of adjusting a microscope, comprising an adjustment step of adjusting the contrast of the observation image by operating one or more.

Images