JP2003131139A - Modulation contrast microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a modulation contrast microscope which optimizes the view of the transparent zone of an egg in implementing an ICSI (intracytoplasmic sperm injection method). SOLUTION: This microscope has a light source 1, a condenser lens 5, an opening 6a arranged in the front side focal position of the condenser lens 5, an objective lens 7 and a modulator 14 which is arranged in a position approximately conjugate with the opening 6a and has a region of transmittance T(%). The microscope is so constituted as to satisfy the conditions attaining 1.05<|(Mo×fc)/Mc×fr)|<1.4, 8<T<25 when Mo is defined as the size in the radial direction of the region of the transmittance T within the surface perpendicular to the optical axis at which the modulator 14 is arranged, Mc as the size in the radial direction of the opening 6a with an optical axis of the microscope as a center, fc as the focal length of the condenser lens 5 and fr as the focal length of the optical system from a specimen surface S to the modular 14.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to microscopes, and in particular to contrast modulated microscopes.

[0002]

2. Description of the Related Art Currently, a microscope is used as one of infertility treatments.
ICSI (intracytoplasmic sperm injection method) is actively used, but in ICSI, the modulation contrast method (Hoffman modulation contrast: a registered trademark of Modulation Optics) is often used. This modulated contrast method
This is a publicly known technique disclosed in Japanese Patent Laid-Open No. 51-29149, and its principle will be briefly described with reference to FIGS. In the figure, 5 is a condenser lens, S is a sample, and 7
Is an objective lens. Further, 6 is an aperture plate, which has a rectangular aperture 6a at a position away from the center. Further, 14 is a disk-shaped modulator, which is arranged at a position substantially conjugate with the aperture plate 6. In this modulator 14, a region 14a having a transmittance of 100%, which can include an image of the aperture 6a, a region 14b having a transmittance of 15%, and a region 14c having a transmittance of 0% are arranged adjacent to each other in this order. There is.

In this optical system, since the rectangular opening 6a is arranged at a position decentered from the optical axis, the light incident on the condenser lens 5 is emitted so as to illuminate the sample S in an oblique direction. At this time, if the transparent sample S is flat as shown in FIG. 6A, the light flux transmitted through the sample S is imaged in the region 14b of the modulator 14 by the objective lens 7,
As shown in (a), an aperture image 6a 'is formed in the area 14b. Further, when the surface of the sample S is a slope that rises to the right as shown in FIG. 6B, the light flux is refracted to the right when passing through the sample S and is formed in the region 14c of the modulator 14. Statue and
As shown in FIG. 7B, an aperture image 6a 'is formed in the area 14c. Further, if the surface of the sample S is an inclined surface that rises to the left as shown in FIG. 6C, the light flux is refracted to the left when passing through the sample S and is imaged in the region 14a of the modulator 14. Then, as shown in FIG. 7C, an aperture image 6a 'is formed in the area 14a.

As is clear from this explanation, the sample S is shown in FIG.
In the case of a colorless transparent body having a flat surface and a slope as shown in (a), the observed image looks gray in the flat surface and black or white in the slope as shown in FIG. 8 (b). As described above, in the modulation contrast method, it is possible to observe a colorless and transparent sample as a stereoscopic image having a shadow due to the different transmittance regions provided in the modulator 14 and the effect of the oblique illumination.

[0005]

The modulated contrast method is used when injecting sperm into an egg cell as described above. In that case, a portion called the zona pellucida at the periphery of the egg, particularly the egg cell and its It is necessary that the boundary with the outside (for example, the culture medium) is clearly visible. Further, in the part inside the peripheral part, it is necessary that the fine structure such as granules present in this part can be seen well. However, the conventional Hoffman modulation contrast has an appropriate contrast when an egg is seen, but has a problem that the fine parts such as the zona pellucida and granules are too high in contrast to be difficult to see.

The present invention has been made in view of the above problems of the prior art, and an object of the present invention is to provide a modulation contrast that optimizes the appearance of the zona pellucida of egg cells and the fine structure inside thereof. To provide a microscope.

[0007]

In order to achieve the above object, a modulation contrast microscope of the present invention comprises a light source, a condenser lens, and an aperture member having a partial aperture arranged in the vicinity of the front focus position of the condenser lens. A modulation contrast microscope including an objective lens and a modulator arranged at a position substantially conjugate with the aperture member, wherein the modulator has a region of transmittance T (%), and satisfies the following conditions. It is characterized by doing so. 1.05 <| (Mo × fc) / Mc × fr) | <1.4 8 <T <25 where Mo is the radial length in the region of the transmittance T, Mc is the radial length in the partial opening, fc is the focal length of the condenser lens, and fr is the focal length of the optical system between the sample surface and the modulator. According to the present invention, the distance D from the optical axis of the microscope in the region of the transmittance T is
It is configured to satisfy the condition of 0.2R <D <0.6R.
However, R is the effective radius of the surface on which the modulator is arranged. Further, the modulation contrast microscope according to the present invention includes a light source, a condenser lens, an aperture member having a partial aperture arranged in the vicinity of the front focus position of the condenser lens, an objective lens, and a position substantially conjugate with the aperture member. A modulator having a modulator arranged therein, wherein the modulator has a region of transmittance T (%), and a plurality of aperture members are provided for one modulator, and 8 <T <25
It is characterized by satisfying the following conditions.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below based on illustrated examples. FIG. 1 is a diagram showing the overall configuration of a modulation contrast microscope according to the present invention. In the figure,
1 is a light source, 2 is a collector lens, 3 is an illumination lens, 4 is a mirror, 5 is a condenser lens, 6 is an aperture plate, 7 is an objective lens, 8 is a revolver, 9, 10, 11, and 12 are relay lenses, and 13 is Eyepiece, 14 modulator, 15 sample
This is a stage for mounting S. The aperture plate 6 is arranged at or near the front focus position of the condenser lens. Then, as shown in FIG. 2, the opening plate 6 is provided with a rectangular opening 6a in the peripheral portion away from the center. In this embodiment, another opening plate 6'having a rectangular opening different from the opening plate 6 can be replaced. The opening plate 6 and the opening plate 6'can be exchanged by, for example, disposing the opening plate 6 and the opening plate 6'in the turret 15 and rotating the turret 15. If you do this,
Either the aperture plate 6 or the aperture plate 6'can be inserted into the optical path. Further, the modulator 14 is arranged at or near the rear focal position of the objective lens 7. This position is a position substantially conjugate with the aperture plate 6 and the aperture plate 6 '.
Further, the modulator 14 is provided with a region 16 having a transmittance of T% as shown in FIG. This area 1 where the transmittance is T%
6 is formed in the peripheral part away from the center like the rectangular opening 6a.

In this embodiment, Mo is the radial length of the region 16 having a transmittance of T%, Mc is the radial length of the rectangular opening 6a, and fc is the condenser lens 5.
The following conditional expression is satisfied, where fr is the focal length of the optical system between the sample surface and the modulator 14. The radial direction is as shown in FIG.
Alternatively, it is in the direction of the arrow shown in FIG. This radial direction is a direction orthogonal to the boundary line showing the change in transmittance in the modulator 14. 1.05 <| (Mo × fc) / (Mc × fr) | <1.4 (1) 8 <T <25 (2)

Since this embodiment is configured as described above, the light from the light source 1 emitted from the rectangular opening 6a (the opening 6b will be described later) is condensed by the condenser lens 5, the sample S and the objective lens 7. And the modulator 14 to form a sample image. This sample image is, as explained in the prior art,
Contrast is provided according to the shape of the sample, and the observer observes through the eyepiece lens 13. Then, the conditional expression (1) is expressed by the size of the region of the transmittance T of the modulator 14 viewed in the radial direction and the radius of the rectangular opening 6a projected in the plane in which the modulator 14 is arranged. The size of the area of the transmittance T is 1.05 times to 1.4 times the size of the aperture image. Since the rectangular opening 6a is arranged near the front focus position of the condenser lens 5, the size of the opening image projected on the modulator 14 in the radial direction is Mc × fr.
It becomes / fc.

In the conventional modulation contrast method, the transmittance T
%, And the size of the rectangular opening projected in this area was almost the same. Therefore, when light passes through a sample having a large change in unevenness (a large change in thickness) like an egg cell, the unevenness causes a large refraction of the light. Therefore, the aperture projected on the modulator 14 is displaced from the region of the transmittance T%. As a result, the contrast becomes too high, and the observed image becomes a glittering image, and fine parts cannot be seen. On the other hand, in the present invention, since the region of the transmittance T% is selected to be larger than the aperture projected on the modulator 14,
The contrast can be suppressed so that fine details can be seen well.

That is, when the value goes below the upper limit of the conditional expression (1), glare becomes too large in an observed image. Therefore, for example, in the case of egg cells, the zona pellucida and the fine parts inside thereof cannot be seen. On the other hand, if this ratio exceeds the upper limit, the contrast becomes too weak and the whole egg cell becomes difficult to see. Further, the conditional expression (2) represents the numerical value condition of the transmittance T%, but when the transmittance falls below the lower limit of the expression (2), for example, in an egg cell, the contrast between the peripheral part and the central part The difference becomes too strong and the image at the center becomes too dark, making it difficult to observe. If this transmittance exceeds the upper limit of the equation (2), the contrast becomes too weak and the whole egg cell becomes difficult to see.

As a result of experiments, it has been found that if | (Mo × fc) / (Mc × fr) | satisfies the following condition, it is possible to perform modulation contrast observation with a better contrast and resolution balance. . 1.15 <| (Mo × fc) / (Mc × fr) | <1.3 (3)

Further, in the present embodiment, when the distance from the optical axis of the microscope in the region of transmittance T% is D and the effective radius of the surface on which the modulator 14 is arranged is R (see FIG. 3), It is preferable that the conditional expression (4) is satisfied. That is, when the value goes below the lower limit of the following expression (4), the region of the transmittance T% is close to the optical axis and the contrast is improved, but the resolution is deteriorated. On the other hand, when the value exceeds the upper limit of the following formula (4), it is advantageous in terms of resolution but the contrast is poor. 0.2R <D <0.6R (4)

In this embodiment, when NAo is the NA of the objective lens having the maximum NA (numerical aperture) and NAc is the NA of the condenser lens 5, it is preferable to satisfy the following conditional expression (5). 0.7NAo <NAc (5) Equation (5) above specifies the NA of the condenser lens, but if this condition is not satisfied, the NA of the condenser lens will be
Becomes small, and it becomes impossible to perform modulation contrast observation with good resolution.

As described above, in the present embodiment, 6a is described as a rectangular opening, but it may be a ring-shaped opening. However, it may be arranged at a position optically conjugate with the rectangular opening 6a on the condenser lens 5 side. The image of the rectangular aperture 6a can be formed inside the objective lens 7 and inside the microscope main body. However, since the number of relays of the aperture image is smaller and the aberration of the aperture image is smaller in the objective lens, the modulator 1
It is desirable that 4 is in the objective lens 7.

The present invention is characterized by observing a modulation contrast microscope with near infrared light. In recent years, in the field of artificial insemination, it is considered that damage to egg cells is less in near infrared light than in visible light, and TV observation by near infrared light has been studied. When using near-infrared light, the image of the rectangular opening 6a projected on the modulator 14 tends to be blurred due to chromatic aberration rather than visible light. Therefore, if the region of the transmittance T is larger than the image of the rectangular opening 6a projected on the modulator 14 as in the formula (1), the TV observation by the near-infrared light is rectangular. The image of the shaped opening 6a does not protrude beyond the region of the transmittance T, and it is possible to perform observation with suppressed contrast.

Further, in the present embodiment, a plurality of aperture members are switched and used for one modulator 14. Regardless of which aperture member is used, the modulator 14 that satisfies the conditional expression (2) is used. As described above, when the rectangular opening 6a is used, the contrast is slightly lowered to suppress the glare and improve the appearance of fine parts. Therefore, when a sample other than egg cells with little unevenness is seen, the contrast may be low and it may be difficult to see. In such a case, switching to the aperture having a large Mc, that is, the aperture member 6 ′ having the rectangular aperture 6b allows observation with good contrast because the lower limit of the formula (1) is not satisfied. .

FIG. 4 is a photograph showing an example of an egg cell image observed by the modulation contrast microscope according to this embodiment.
(A) is an overall view, and (b) is an enlarged view of the edge portion of the egg cell.
The magnification of the objective lens used is 20X, NA is 0.4, NA of condenser lens is 0.5, | (Mo × fc) / Mc × fr) | = 1.2, T = 11
%, D / R = 0.53.

FIG. 5 is a photograph showing an example of an egg cell image observed by a conventional modulation contrast microscope. (A) is an overall view and (b) is an enlarged view of the edge portion of the egg cell. The magnification of the used objective lens is 20X, NA is 0.4, NA of the condenser lens is 0.5, | (Mo × fc) / Mc × fr) | = 1, T = 15%, D / R =
It is 0.625.

In FIGS. 4 (a) and 5 (a), the egg cell has a circular portion at the center and a ring-shaped (ring-shaped) portion around the circular portion.
It is divided into parts. Of these, the ring-shaped part is the transparent band. Here, FIG. 4B and FIG. 5B are compared. In each figure, a ring-shaped transparent band and the structure inside thereof are shown. Here, in FIG. 5B, the boundary between the transparent band and its inner structure is shining white, and the boundary between the transparent band and the outside is black. On the other hand, in FIG. 4B, the boundary between the transparent band and its inner structure, and the boundary between the transparent band and the outside are almost gray.
Therefore, the boundary of the transparent band can be recognized better in FIG. Further, the fine granular structure in the peripheral portion of the internal structure of the circular portion can be better observed in FIG. 4 (b).
As is clear from the comparison between FIG. 4 (b) and FIG. 5 (b), according to the present invention, the edge portion of the egg cell can be observed much more clearly than in the case of the conventional modulation contrast.

As described above, the modulation contrast microscope of the present invention has the following features in addition to the features described in the claims. (1) The modulator according to claim 1, further comprising a modulator arranged at a position substantially conjugate with the aperture and having a region of transmittance T (%) on which an image of the aperture is projected, and the condition below is satisfied. Contrast microscope. 1.15 <| (Mo × fc) / Mc × fr) | <1.3 (2) The modulated contrast microscope according to claim 1, which satisfies the following condition. 0.7NAo <NAc where NAo is the NA of the objective lens for which the NA of the modulation contrast microscope is the maximum, and NAc is the NA of the condenser lens. (3) The modulated contrast microscope according to claim 1, wherein the opening has a rectangular shape. (4) The modulation contrast microscope according to claim 1, wherein the modulator is arranged in the objective lens. (5) The modulated contrast microscope according to claim 1, which is configured to be observed using near infrared light.

[0023]

As described above, according to the present invention, it is possible to provide a modulated contrast microscope in which the appearance of the transparent band is optimized during ICSI.

[Brief description of drawings]

FIG. 1 is a diagram showing an overall configuration of a modulation contrast microscope according to the present invention.

FIG. 2 is a plan view of an aperture plate used in the modulation contrast microscope according to the present invention.

FIG. 3 is a plan view of a modulator used in the modulation contrast microscope according to the present invention.

4A and 4B are photographs showing an example of an egg cell image observed by a modulation contrast microscope according to an example of the present invention, where FIG. 4A is an overall view and FIG. 4B is an enlarged view of an edge portion of an egg cell.

FIG. 5 is a photograph showing an example of an egg cell image observed by a conventional modulation contrast microscope, (a) is an overall view, (b)
[Fig. 3] is an enlarged view of an edge portion of an egg cell.

FIG. 6 is a diagram for explaining the principle of modulation contrast.

FIG. 7 is a diagram for explaining the positional relationship between the aperture image and the modulator, where (a), (b), and (c) correspond to (a), (b), and (c) of FIG. 6, respectively. is doing.

FIG. 8A is a diagram showing an example of the shape of a sample, and FIG. 8B is a diagram showing shades appearing corresponding to this sample.

[Explanation of symbols]

1 light source 2 collector lens 3 illumination lens 4 mirror 5 condenser lens 6 aperture plates 6a, 6b apertures 6a ', 6b' aperture image 7 objective lens 8 revolver 9, 10, 11, 12 relay lens 13 eyepiece lens 14 modulator S sample

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 2H052 AA06 AC05 AC13 AC28 AC29                       AD03 AD34 AF19                 4C060 HH01

Claims (3)

[Claims] 1. A light source, a condenser lens, an aperture member having a partial aperture disposed in the vicinity of a front focal position of the condenser lens, an objective lens, and a modulator disposed at a position substantially conjugate with the aperture member. And a modulator having a region of transmittance T (%) so that the following condition is satisfied. 1.05 <| (Mo × fc) / Mc × fr) | <1.4 8 <T <25 where Mo is the radial length in the region of the transmittance T and Mc is the radial length fc in the partial opening. Is the focal length of the condenser lens, and fr is the focal length of the optical system between the sample surface and the modulator. 2. The microscope according to claim 1, wherein the distance D of the region of the transmittance T from the optical axis of the microscope satisfies the following condition. 0.2R <D <0.6R where R is the effective radius of the surface on which the modulator is placed. 3. A light source, a condenser lens, an aperture member having a partial aperture disposed in the vicinity of a front focal position of the condenser lens, an objective lens, and a modulator disposed at a position substantially conjugate with the aperture member. And a modulator having a region of transmittance T (%), and a plurality of aperture members are provided for one modulator so that the following conditions are satisfied. A modulated contrast microscope characterized in that 8 <T <25