U.S. patent application number 13/334256 was filed with the patent office on 2012-04-19 for microscope system.
This patent application is currently assigned to NIKON CORPORATION. Invention is credited to KUMIKO MATSUI, Katsuya WATANABE.
Application Number | 20120092761 13/334256 |
Document ID | / |
Family ID | 40901038 |
Filed Date | 2012-04-19 |
United States Patent
Application |
20120092761 |
Kind Code |
A1 |
MATSUI; KUMIKO ; et
al. |
April 19, 2012 |
MICROSCOPE SYSTEM
Abstract
A microscope system is characterized in comprising a
transmission illumination optical system having a light source (11)
and a condenser lens (13); a first dry objective (15a) having a
magnification of from 20 or higher to 40 or lower and capable of
viewing by at least one of a differential interference viewing
method and a modulation contrast viewing method; and a second dry
objective (15b) having a magnification of from 60 or higher to 100
or lower and capable of viewing by a differential interference
viewing method; the first objective (15a) and the second objective
(15b) being exchangeable.
Inventors: |
MATSUI; KUMIKO;
(Yokohama-shi, JP) ; WATANABE; Katsuya;
(Yokohama-shi, JP) |
Assignee: |
NIKON CORPORATION
|
Family ID: |
40901038 |
Appl. No.: |
13/334256 |
Filed: |
December 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12840248 |
Jul 20, 2010 |
8098427 |
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13334256 |
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PCT/JP2009/050555 |
Jan 16, 2009 |
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12840248 |
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Current U.S.
Class: |
359/370 |
Current CPC
Class: |
Y10S 359/90 20130101;
A61B 2090/373 20160201; G02B 21/14 20130101; G02B 21/02 20130101;
G02B 13/22 20130101; A61B 90/20 20160201 |
Class at
Publication: |
359/370 |
International
Class: |
G02B 21/02 20060101
G02B021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 23, 2008 |
JP |
2008-012823 |
Claims
1-8. (canceled)
9. A microscope system for micro-insemination, comprising: a
transmission illumination optical system having a light source and
a condenser lens; a first dry objective having a magnification of
from 20 or higher to 40 or lower and capable of viewing by at least
one of a differential interference viewing method and a modulation
contrast viewing method; and a second dry objective having a
magnification of from 60 or higher to 100 or lower and capable of
viewing by a differential interference viewing method; the first
objective and the second objective being exchangeable.
10. The microscope system for micro-insemination according to claim
9, characterized in that the following conditional expressions are
satisfied: 0.78.ltoreq.NA<1.0 f/3.ltoreq.WD<2f, where NA is
the numerical aperture of the second objective, f is focal length
thereof, and WD is working distance thereof.
11. The microscope system for micro-insemination according to claim
9, characterized in that the following conditional expression is
satisfied when viewing by the differential interference viewing
method using the second objective:
0.3.lamda./NA.ltoreq.S.ltoreq.0.61.lamda./NA, where S is the shear
distance in the object plane, NA is the numerical aperture of the
second objective, and .lamda. is the wavelength of the viewed
light.
12. The microscope system for micro-insemination according to claim
9, characterized in that the second objective has a correction ring
for correcting aberration fluctuation due to changes in factors
including temperature and cover glass thickness.
Description
[0001] This is a continuation of PCT International Application No.
PCT/JP2009/050555, filed on Jan. 16, 2009, which is hereby
incorporated by reference. This application also claims the benefit
of Japanese Patent Application No. 2008-012823, filed in Japan on
Jan. 23, 2008, which is hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a microscope system for use
with micro-insemination, the system being capable of viewing using
a differential interference viewing method and a modulation
contrast viewing method.
TECHNICAL BACKGROUND
[0003] At present, ICSI (intra-cytoplasmic sperm injection) is
widely used as a micro-insemination method. In micro-insemination,
a sperm is selected using a modulation contrast viewing method (see
Patent Document 1, for example), and a sperm having satisfactory
motility and morphology is injected into an ovum. However, recent
advances in IVF (in-vitro fertilization) research have shown
statistically that such factors as the presence, size, and number
of vacuoles in the sperm head are significantly related to the IVF
success rate, but vacuoles in the sperm head are difficult to view
by the modulation contrast viewing method used for conventional
ICSI. Therefore, a microscope system has been proposed for enabling
IMSI (intra-cytoplasmic morphologically selected sperm injection,
which is a micro-insemination method in which a sperm is selected
under high magnification), in which micro-insemination is performed
after selection by detailed viewing of the inside of the sperm
head, to be performed in addition to the conventional ICSI. For
example, a configuration is adopted in which the modulation
contrast viewing method used in ICSI is employed jointly with a
differential interference viewing method (see Patent Documents 2
and 3, for example) through a high-magnification objective that is
used in IMSI.
[0004] Patent Document 1: Japanese Laid-open Patent Publication No.
S51-128548
[0005] Patent Document 2: Japanese Patent No. 3456252
[0006] Patent Document 3: Japanese Patent No. 3415294
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0007] In the differential interference viewing method used in the
micro-insemination described above and well as in other fields of
biological microscopy, structures must be observable in as much
detail as possible, and an immersion objective having a high
numerical aperture (NA) has generally been used as a
high-magnification lens. As a result, in the conventional
microscope system, an immersion-type lens is used as a
high-magnification objective, and a dry-type lens is used as a
medium-low-magnification objective, and during the switch to the
dry-type medium-low-magnification objective for ICSI viewing after
IMSI viewing through the immersion-type high-magnification
objective, the immersion liquid significantly interferes with the
workability of IVF. In order to overcome this problem, a system has
been proposed in which an immersion objective is used as the
medium-low-magnification objective for ICSI viewing, as with the
high-magnification objective. In this system, however, the
viscosity of the immersion liquid causes the sample (usually a
dish) to move when immersion objectives are used exchangeably, the
sperm selected using the high-magnification objective may move out
of view, and bubbles and the like are prone to be introduced into
the immersion liquid. These problems can make ICSI viewing
extremely difficult after the objectives are exchanged.
[0008] The present invention was developed in view of such
problems, and an object of the present invention is to provide a
microscope system suitable for IMSI/ICSI, whereby the sequence of
operations for micro-insemination can be accurately and rapidly
performed while maintaining resolving power, by viewing and
selecting a sperm by a differential interference viewing method
using a dry-type high-magnification objective, then injecting the
selected sperm into an ovum by a differential interference viewing
method or a modulation contrast viewing method using an exchanged
low-magnification objective, which is also a dry-type
objective.
Means to Solve the Problems
[0009] In order to achieve such objects as those described above,
the present invention is a microscope system suitable for
micro-insemination, and is characterized in comprising a
transmission illumination optical system having a light source and
a condenser lens; a first dry objective having a magnification of
from 20 or higher to 40 or lower and capable of viewing by at least
one of a differential interference viewing method and a modulation
contrast viewing method; and a second dry objective having a
magnification of from 60 or higher to 100 or lower and capable of
viewing by a differential interference viewing method; the first
objective and the second objective being exchangeable.
Advantageous Effects of the Invention
[0010] As described above, according to the present invention, a
microscope system suitable for micro-insemination can be provided
whereby the sequence of operations in micro-insemination can be
rapidly and accurately performed with satisfactory workability
while the appropriate resolving power is maintained.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic sectional view showing the microscope
system utilizing a differential interference viewing method
according to the present embodiment;
[0012] FIG. 2 is a view showing the underlying principle of the
modulation contrast viewing method, which is another viewing method
used in the microscope system of the present embodiment;
[0013] FIG. 3 is a view showing the positional relationship between
the aperture image and the modulator in the modulation contrast
viewing method according to the present embodiment, wherein FIG.
3A, FIG. 3B, and FIG. 3C correspond to FIG. 2A, FIG. 2B, and FIG.
2C;
[0014] FIG. 4A is a view showing an example of the shape of the
sample, and FIG. 43 is a view showing the shading that appears
corresponding to the sample in the modulation contrast viewing
method according to the present embodiment;
[0015] FIG. 5 is a view showing the MTF curve of the incoherent
optical system according to the present embodiment;
[0016] FIG. 6 is a view showing the contrast MTF curves of phase
samples in the differential interference viewing method according
to the present embodiment;
[0017] FIG. 7 is a sectional view showing the structure of the
second objective (dry-type high-magnification objective) according
to a first example; and
[0018] FIG. 8 shows several aberration diagrams for the second
objective according to the first example, wherein FIG. 8A is a
spherical aberration diagram, FIG. 8B is an astigmatism diagram,
and
[0019] FIG. 8C is a distortion diagram.
EXPLANATION OF NUMERALS AND CHARACTERS
[0020] 11 light source (transmission illumination optical system)
[0021] 12 collector lens [0022] 13 condenser lens (transmission
illumination optical system) [0023] 14 sample [0024] 15 objective
[0025] 15a medium-magnification objective (first objective)
(capable of viewing by a differential interference viewing method)
[0026] 15b high-magnification objective (second objective) [0027]
16 turret [0028] BP1 illumination-side birefringent optical member
[0029] BP2 imaging-side birefringent optical member [0030] P
polarizer [0031] A analyzer [0032] 22 medium-magnification
objective (first objective) (capable of viewing by a modulation
contrast viewing method) [0033] 23 aperture plate [0034] 23a
rectangular aperture [0035] 24 modulator
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] Preferred embodiments will be described with reference to
the drawings. FIG. 1 is a schematic sectional view showing a
microscope system suitable primarily for micro-insemination,
according to the present embodiment. The microscope system
according to the present embodiment is suitable for IMSI
(intra-cytoplasmic morphologically selected sperm injection, which
is a micro-insemination method in which a sperm is selected under
high magnification) and ICSI (intra-cytoplasmic sperm injection),
and as shown in FIG. 1, the microscope system has a transmission
illumination optical system composed of a light source 11 and a
condenser lens 13; a collector lens 12; a sample 14; an objective
15; a turret 16; an illumination-side birefringent optical member
BP1; an imaging-side birefringent optical member BP2; a polarizer
P; and an analyzer A.
[0037] In FIG. 1, the illuminating light from the light source 11
is incident on the polarizer P after being collected by the
collector lens 12, and is converted to linearly polarized light.
The illumination-side birefringent optical member BP1 and the
condenser lens 13 for irradiating the illuminating light on the
sample 14 are provided in order from the light source 11 side in
the optical path between the polarizer P and the sample 14. The
linearly polarized light emitted from the polarizer P is incident
on the illumination-side birefringent optical member BP1 and split
by birefringence into two linearly polarized light components
having mutually orthogonal directions of vibration, and the
linearly polarized light components are incident on the condenser
lens 13. The two rays split by the illumination-side birefringent
optical member BP1 travel at a small separation angle .alpha., are
converted to parallel rays separated from each other at a small
shear distance S by the collecting effect of the condenser lens 13,
and illuminate the sample 14. The two rays transmitted at slightly
separated positions on the sample 14 are incident on the
imaging-side birefringent optical member BP2 via the objective 15
and combined by the birefringent effect of the imaging-side
birefringent optical member BP2 so as to travel on the same optical
path. The combined rays are incident on the analyzer A, the
analyzer A extracts only the components of the mutually orthogonal
linearly polarized light that are vibrating in the same direction,
and these components interfere. As a result, a magnified image
(interference image 17) on the image plane is formed by an
interference fringe that forms according to the phase difference
imparted between the two light rays as the rays pass through the
sample 14 in slightly different positions. An observer can view the
magnified image 17 through an eyepiece optical system not shown in
the drawing.
[0038] When the sample 14 is planar and homogeneous, (since there
is no phase difference between the two split rays of light,) the
magnified image 17 is an image that has a uniform intensity
distribution and is devoid of contrast. On the other hand, when the
sample 14 is heterogeneous and has gradients and level differences,
(since there is a phase difference between the two split rays of
light,) contrasts occur in the magnified image 17 in portions where
the refractive index varies, or in portions that correspond to
gradients and level differences. The refractive index variations or
the gradients and level differences are thereby made visible, and
the sample 14 can be viewed at magnification.
[0039] The objective 15 is composed of a first objective 15a having
a magnification of from 20 or higher to 40 or lower (hereinafter
referred to as a dry-type medium-magnification objective or a
medium-magnification objective), and a second objective 15b having
a magnification of from 60 or higher to 100 or lower that is
capable of contrast viewing by a differential interference viewing
method (hereinafter referred to as a dry-type high-magnification
objective or a high-magnification objective), and the
medium-magnification objective 15a and the high-magnification
objective 15b are configured so as to be exchangeable with the aid
of a turret 16 or the like.
[0040] The first objective 15a is preferably capable of viewing by
at least one of the abovementioned differential interference
viewing method or modulation contrast viewing method. The
underlying principles of the modulation contrast viewing method
will be briefly described using FIGS. 2 through 4. In the drawings,
the reference numeral 21 refers to a condenser lens, S refers to a
sample, 22 refers to an objective, 23 refers to an aperture plate,
and 24 refers to a disk-shaped modulator. The aperture plate 23 has
a rectangular aperture 23a positioned at a distance from the
center, in the focal position on the light source side of the
condenser lens 21. The modulator 24 is provided in a position
substantially conjugate to the aperture plate 23, and a 100%
transmittance region 24a that may include the image of the aperture
23a, a region 24b of 15% transmittance, for example, and a 0%
transmittance region 24c are formed in the stated order adjacent to
each other in the modulator 24.
[0041] In this optical system, since the rectangular aperture 23a
is disposed in an eccentric position with respect to the optical
axis, light that is incident on the condenser lens 21 is emitted so
as to illuminate the sample S at an oblique angle. When the
transparent sample S is planar as shown in FIG. 2A, the flux of
light passing through the sample S is focused in the region 24b of
the modulator 24 by the objective 22, and an aperture image 23a' is
formed in the region 24b, as shown in FIG. 3A. When the surface of
the sample S is inclined so as to rise to the right, as shown in
FIG. 2B, the flux of light that passes through the sample S is
refracted to the right and focused in the region 24c of the
modulator 24, and the aperture image 23a' is formed in the region
24c, as shown in FIG. 3B. When the surface of the sample S is
inclined so as to rise to the left, as shown in FIG. 2C, the flux
of light that passes through the sample S is refracted to the left
and focused in the region 24a of the modulator 24, and the aperture
image 23a' is formed in the region 24a, as shown in FIG. 3C.
[0042] As is apparent from this description, when the sample S is a
colorless transparent body having flat surfaces and inclined
surfaces such as shown in FIG. 4A, the viewed image is such that
the flat portions appear gray and the inclined portions appear
black or white such as shown in FIG. 4B. The modulation contrast
viewing method thus enables even a colorless transparent sample to
be viewed as a three-dimensional image with shading, through the
effects of focal illumination and regions of the modulator 24
having different transmittances.
[0043] In this microscope system, when the medium-magnification
objective 15a that is capable of contrast viewing by a modulation
contrast viewing method is used, a change is made to the
illumination-side birefringent optical member BP1, imaging-side
birefringent optical member BP2, polarizer P, and analyzer A, which
are members used for viewing by the differential interference
viewing method described above; and the aperture plate 23 and the
modulator 24 are placed between the light source 11 and the
condenser lens 13 (in the stated order from the light source
11).
[0044] In the microscope system according to the present
embodiment, the following Conditional Expressions (1) and (2) are
preferably satisfied, where NA is the numerical aperture of the
dry-type second objective (high-magnification objective), f is the
focal distance, and MD is the working distance.
0.78.ltoreq.NA<1.0 (1)
f/3.ltoreq.WD<2f (2)
[0045] In viewing by the differential interference viewing method
using the second objective, the following Conditional Expression
(3) is preferably satisfied, where S is the shear distance in the
object plane, NA is the numerical aperture of the second objective,
and .lamda. is the wavelength of the viewed light.
0.3.ltoreq.S.ltoreq.0.61.lamda./NA (3)
[0046] In the past, resolving ability was considered critical in
viewing by the differential interference viewing method using a
high-magnification (60 or higher and 100 or lower), high numerical
aperture immersion objective in the field of biological microscopy,
and intervention using video enhancement or other image processing
was assumed. However, during IMSI or other micro-insemination,
visual observation is consistently used in order to rapidly and
accurately perform the sequence of operations whereby a more
satisfactory sperm is selected from a wide range of (numerous)
sperm under high magnification, and the selected sperm is then
injected into an ovum under medium magnification. There is
accordingly a need for visually adequate contrast, but optimum
conditions have not yet been presented for an objective capable of
viewing by a differential interference viewing method that would
satisfy the need for adequate contrast. The optimum Conditional
Expressions (1) through (3) have therefore been derived for the
second objective (high-magnification objective) for enabling
viewing by the differential interference viewing method in the
present microscope system. There follows a description of
Conditional Expressions (1) through (3) in the stated order.
[0047] Conditional Expression (1) for specifying the range of
appropriate numerical apertures NA of the high-magnification
objective will first be described. In the present microscope
system, the head portion of a sperm, an image of which is viewed in
the present microscope system, is about 4 to 5 .mu.m in size, and
it has been confirmed that one to ten vacuoles of various sizes are
scattered through the head portion in a single focal plane.
Consequently, the ability to see and identify details about 0.4 to
0.5 .mu.m in size with good contrast is understood to be adequate
for IMSI applications.
[0048] FIG. 5 shows the MTF curve (i.e., the relationship between
contrast on the vertical axis and the resolving power of the
optical system on the horizontal axis) of the incoherent optical
system commonly used in a microscope optical system. On the
horizontal axis f/f0 of FIG. 5, the spatial frequency f is
normalized so that f0=NA/.lamda., where f is the spatial frequency,
NA is the numerical aperture of the objective, and .lamda. is the
wavelength of the viewed light. The relationship to the horizontal
resolving power RES corresponding to the spatial frequency f is
indicated by Equation (4), where RES is the horizontal resolving
power of the microscope optical system.
RES=1/f={1/(f/f0)}.times.{.lamda./NA} (4)
[0049] The maximum value of 2.0 for the horizontal axis f/f0 shown
in FIG. 5 corresponds to the maximum resolving power RES(max) of
the microscope optical system. Therefore, by substituting f/f0=2.0
in Equation (4), Equation (5) is obtained, in which the maximum
resolving power RES(max) of the microscope optical system is
indicated. An image is difficult to see when there is no additional
margin beyond the numerical aperture NA that corresponds to the
maximum resolving power RES(max) specified by Conditional
Expression (5).
RES(max)={1/2.0}.times.{.lamda./NA}=0.5.lamda./NA (5)
[0050] It is known that the human eye generally has difficulty
distinguishing contrast values of 0.1 or lower, and that visibility
is satisfactory when the contrast value is 0.2 or higher. In FIG.
5, since the spatial frequency f/f0 at which the contrast value is
0.1 is near 1.6, Equation (6) must be satisfied by substituting
these values into Equation (4) in order for the horizontal
resolving power RES to be 0.4 .mu.m (the size of a vacuole in the
sperm head as the viewed image).
RES={1/1.6}.times.{.lamda./NA}=0.4 (6)
[0051] Since visual observation is assumed in IMSI and ICSI, which
are the applications for which the present microscope system is
used, the central wavelength .lamda. during viewing is preferably
in the vicinity of 500 nm to 550 nm when visibility to the eye is
considered. The reason for this is that 500 nm and 550 nm are
visibility peaks for dark locations and bright locations,
respectively. When the central wavelength .lamda. of 500 nm=0.5
.mu.m for viewing is substituted into Equation (6), Equation (7)
below is obtained as the lower limit of the numerical aperture NA
required for the high-magnification objective.
NA={1/1.6}.times.0.5 [.mu.m]/0.4 [.mu.m]=0.78 (7)
[0052] Since the spatial frequency f/f0 at which the contrast value
is 0.2, which is considered to produce good visibility, is near 1.4
according to FIG. 5, a more preferred lower limit for the numerical
aperture NA is obtained from Equation (8) by substituting into
Equation (6) in the manner described above.
NA={1/1.4}.times.0.5 [.mu.m]/0.4 [.mu.m]=0.89 (8)
[0053] The numerical aperture NA is indicated by NA=nsin(.phi./2),
where n is the refractive index of the medium between the objective
and the sample, and .phi. is the aperture angle, and the medium in
the present embodiment is air (refractive index n=1). Therefore,
the maximum numerical aperture NA is 1.
[0054] In summary, in the microscope system of the present
embodiment, the range of the numerical aperture NA of the
high-magnification objective preferred for enabling visual
observation with good contrast is expressed by Conditional
Expression (1), i.e., 0.78.ltoreq.NA<1.0. The range of the
numerical aperture NA of the high-magnification objective is more
preferably 0.89.ltoreq.NA<1.0, according to Conditional
Expression (8).
[0055] Conditional Expression (2) will next be described, which
specifies the appropriate range of the working distance WD in the
high-magnification objective. In the conventional microscope
system, a large working distance is generally difficult to obtain
with the high-numerical-aperture immersion objective used as the
high-magnification objective. Therefore, when such an objective is
used in ICSI viewing or IMSI viewing, which require that the sample
be kept at 37.degree. C. by an insulating device or the like during
working, the insulation device and the distal end of the objective
are prone to interfere during the switch from high magnification to
the medium-magnification objective. Since a large working distance
cannot be obtained, only the area immediately below the cover glass
can be viewed by the high-numerical-aperture immersion objective,
and sperm that are positioned at a distance from the cover glass
cannot be targeted for selection. Therefore, a dry-type lens is
used as the high-magnification objective in the present microscope
system, and the condition of having the large working distance WD
indicated by Conditional Expression (2) is thereby provided in
addition to the condition of the numerical aperture NA indicated by
Conditional Expression (1).
[0056] One factor that determines the working distance is the
magnification of the objective. In an infinity-corrected optical
system, the magnification of the objective is determined by the
ratio of the focal distances of the imaging lens and the objective
(in a high-magnification objective having a magnification of from
60 or higher to 100 or lower such as in the present embodiment,
when the focal distance of the imaging lens is 200 .mu.m, for
example, the focal distance of the objective is 2.00 to 3.33 m),
and the focal distance shortens as the magnification increases. In
general, since the working distance is proportional to the focal
distance of the objective, the working distance decreases as the
magnification of the objective increases. Another factor that
determines the working distance is the numerical aperture. When the
size of the numerical aperture is the same, a longer working
distance corresponds to a greater height of the light rays at the
first lens surface (surface of the lens on the object side) of the
objective, and aberration becomes more difficult to correct.
[0057] In other words, since increasing the working distance of the
objective is incompatible with increasing the magnification and
numerical aperture thereof, high-magnification objectives used in
conventional microscope systems are polarized between those with an
emphasis on working distance and those with an emphasis on
magnification and numerical aperture. Specifically, magnification
60/numerical aperture 0.7/working distance 2 mm are typical
specifications for working-distance-oriented objectives; and
magnification 100/numerical aperture 1.4/working distance 0.1 mm
are typical specifications for
magnification/numerical-aperture-oriented objectives. However, when
viewing IMSI is the intended application, the numerical aperture NA
does not necessarily exceed 1, as indicated by Conditional
Expression (1), and increasing the working distance by a
corresponding amount leads to enhanced working efficiency.
[0058] Therefore, Conditional Expression (2), i.e.,
f/3.ltoreq.WD<2f, is preferably satisfied in the present
embodiment, where NA is the numerical aperture of the
high-magnification objective (second objective), f is the focal
distance, and WD is the working distance. When Conditional
Expression (2) is below the lower limit value, there is increased
risk of such problems as interference between the distal end of the
objective and the insulation device during exchanging of the
objectives. When Conditional Expression (2) exceeds the upper limit
value, the light rays at the first lens surface of the objective
are too high, and it is difficult to ensure the numerical aperture
NA specified by Conditional Expression (1).
[0059] Conditional Expression (3) will next be described.
Conditional Expression (3) specifies the optimum range of the shear
distance S in the differential interference viewing method. FIG. 6
shows the phase contrast MTF curves as the shear distance S is
varied from 1.5.lamda./NA to 0.15.lamda./NA (Patent Document 3
gives a detailed description of the method for computing the phase
contrast MTF in the differential interference viewing method). In
FIG. 6, on the horizontal axis f/f0, the spatial frequency f is
normalized by the reference frequency f0=NA/.lamda. specified by
the numerical aperture NA of the objective, and the vertical axis
indicates the contrast MTF for the phase object at each
frequency.
[0060] It is apparent from FIG. 6 that the phase contrast MTF
curves in the differential interference viewing method show larger
contrast values than the incoherent MTF curve. It is also apparent
from FIG. 6 that the phase contrast MTF has a negative value when
the shear distance S is large (e.g., 1.5.lamda./NA), and this
negative value indicates a state referred to as spurious
resolution, in which black and white are inverted. A state in which
spurious resolution does not occur, i.e., a state in which the
phase contrast MTF is not negative, is generally considered to be
preferable in viewing by the differential interference viewing
method.
[0061] Therefore, the limits of a low spatial frequency band
(region in which the value of the horizontal axis f/f0 is small)
that satisfies a condition whereby the phase contrast MTF value is
not negative will first be described. It is apparent from FIG. 6
that the maximum shear distance S of the low spatial frequency band
is approximately 0.61.lamda./NA to 0.5.lamda./NA, and these values
correspond precisely with the point resolving power or line
resolving power of the microscope optical system. The curve for a
shear distance S of 0.61.lamda./NA in FIG. 6 shows that the
contrast values are high in the low spatial frequency band, but
that the contrast value is about 0.1 in the vicinity of horizontal
axis f/f0=1.4 in the high spatial frequency band, corresponding
precisely with the limit of visibility. The maximum value of the
shear distance S of the objective in the present embodiment is thus
0.61.lamda./NA, at which the point resolving power can be
maintained. A more preferred maximum value for the shear distance S
is 0.5.lamda./NA, (which is closer to the incoherent MTF curve than
the curve for 0.61.lamda./NA,) at which the line resolving power
can be maintained.
[0062] The limits of the high spatial frequency band (region in
which the value of the horizontal axis f/f0 is large) that
satisfies the condition whereby the phase contrast MTF is not
negative will next be described. It is apparent from FIG. 6 that
the maximum shear distance S of the high spatial frequency band
corresponds to the curve having the highest contrast in the
vicinity of horizontal axis f/f0=1.6, i.e., the curve for
0.3.lamda./NA. In this instance, instead of sacrificing contrast in
the low spatial frequency band to a certain degree, the visibility
in the high spatial frequency band can be kept at 0.1, which is
substantially equal to the contrast value of the incoherent MTF.
When the shear distance S is further reduced, contrast decreases in
the low spatial frequency band as well as in the high spatial
frequency band, and is unsuitable for the purposes of the present
embodiment. The minimum value of the shear distance S for the
objective in the present embodiment is thus 0.32.lamda./NA.
[0063] In summary, by setting a shear distance S that satisfies
Conditional Expression (3), i.e.,
0.3.lamda./NA.ltoreq.S.ltoreq.0.61.lamda./NA, and more preferably
0.32.lamda./NA.ltoreq.S.ltoreq.0.5.lamda./NA, vacuoles in the sperm
head can be visualized with high contrast and with almost no
compromise to the resolving power of the objective in a
differential interference viewing method.
[0064] The second objective (high-magnification objective)
according to the present embodiment preferably has a correction
ring for correcting aberration fluctuation due to changes in
temperature, cover glass thickness, and other factors. This is
because the use of a correction ring makes it possible to eliminate
aberration caused by temperature, error in the cover glass
thickness, and other factors; and to make adjustments so that the
resolution and contrast of the objective are both maximized.
EXAMPLES
[0065] Examples of the second objective (dry-type
high-magnification objective) according to the present embodiment
will be described.
First Example
[0066] A first example will be described using FIG. 7, FIG. 8, and
Table 1. FIG. 7 is a sectional view showing the lens structure of
the second objective (dry-type high-magnification lens) according
to the present example. As shown in FIG. 7, the microscope
objective in the present example comprises, in order from the
object, a positive meniscus lens L1 having a concave surface facing
the object; a positive meniscus lens L2 having a concave surface
facing the object; a cemented lens composed of a double-concave
lens L3 and a double-convex lens L4; a cemented lens composed of a
double-concave lens L5 and a double-convex lens L6; a cemented lens
composed of a planoconcave lens L7 and a double-convex lens L8; a
double-convex lens L9; a cemented lens composed of a negative
meniscus lens L10 having a concave surface facing the object, a
double-convex lens L11, and a negative meniscus lens L12 having a
concave surface facing the object; a cemented lens composed of a
double-convex lens L13 and a double-concave lens L14; and a
cemented lens composed of a double-concave lens L15 and a
double-convex lens L16. A cover glass C is provided on the object
side of the positive meniscus lens L1.
[0067] Table 1 shows the various values of the lenses that
constitute the second objective of the present example. In the
various entries shown in Table l, m represents the order of lens
surfaces (hereinafter referred to as surface numbers) from the
object along the direction of travel of a ray of light, r
represents the radius of curvature of each lens, d represents the
distance on the optical axis from each optical surface to the next
optical surface (or image surface), nd represents the refractive
index with respect to the d-line (wavelength: 587.6 nm), and .nu.d
represents the Abbe number based on the d-line. Surface numbers 1
through 25 in Table 1 correspond to surfaces 1 through 25 shown in
FIG. 7. In the table, .beta. represents the magnification, WD
represents the working distance, and NA represents the numerical
aperture.
[0068] In the table, the radius of curvature r, the distance d to
the next lens surface, and other lengths are generally represented
in millimeter units. However, since equivalent optical performance
is obtained whether in proportional magnification or proportional
reduction in the optical system, the units are not limited to
millimeters; other appropriate units may be used. The value
".infin." for the radius of curvature in the table indicates a
plane, and the refractive index of "1.00000" for air is not
noted.
TABLE-US-00001 TABLE 1 [Lens data] .beta. = 100, WD = 1.4, NA =
0.85 m r d nd .nu.d .infin. 0.17000 1.52216 58.80 (cover glass C)
.infin. 2.50462 1 -6.47161 2.37000 1.81600 46.621 2 -4.72849
0.10000 3 -83.0402 2.83000 1.49782 82.557 4 -10.6607 0.15000 5
-46.5266 1.00000 1.61340 44.266 6 24.27074 4.95000 1.43385 95.247 7
-14.7782 0.20000 8 -174.834 1.00000 1.61340 44.266 9 24.11495
4.95000 1.43385 95.247 10 -14.5394 0.20000 11 .infin. 1.00000
1.61340 44.266 12 28.67355 4.20000 1.43385 95.247 13 -23.0153
0.20000 14 48.93548 3.00000 1.49782 82.557 15 -65.8669 1.52002 16
21.78198 1.00000 1.72916 54.660 17 11.99437 6.30000 1.49782 82.557
18 -12.5334 1.20000 1.75500 52.318 19 -59.9845 7.75003 20 27.89895
3.35000 1.59240 68.328 21 -7.03528 8.40000 1.65412 39.682 22
5.87805 1.40000 23 -4.44814 1.00000 1.80440 39.567 24 11.0118
1.90000 1.92286 18.896 25 -11.4804
[0069] FIG. 8 shows several aberration diagrams for the microscope
objective according to the present example, wherein FIG. 8A is a
spherical aberration diagram, FIG. 8B is an astigmatism diagram,
and
[0070] FIG. 8C is a distortion diagram. In FIG. 8, NA is the
numerical aperture, y is the image height (mm), the solid line is
the d-line (wavelength: 587.6 nm), the dashed line is the C-line
(wavelength: 656.3 nm), the single-dashed line is the F-line
(wavelength: 486.1 nm), and the double-dashed line is the g-line
(wavelength 435.8 nm). In the astigmatism diagram, the solid line
represents the sagittal image surface, and the dashed line
represents the meridional image surface.
[0071] As is apparent from the aberration diagrams shown in FIG. 8,
aberrations are satisfactory corrected, and excellent imaging
performance is maintained in the second objective (dry-type
high-magnification objective) according to the present example.
[0072] As described above, according to the present invention,
there is provided a microscope system suitable for IMSI/ICSI,
whereby it is possible to accurately and rapidly perform the
sequence of operations in which the presence of vacuoles in a sperm
head and other characteristics are viewed by a differential
interference viewing method using a dry-type high-magnification (60
or higher and 100 or lower) objective to select a sperm, whereupon
the objectives are exchanged through the use of the turret 16 or
the like, and the selected sperm is injected into an ovum while
viewed by a differential interference viewing method or modulation
contrast viewing method using a medium-magnification (20 or higher
and 40 or lower) objective, which is also a dry-type objective.
[0073] The essential characteristics of embodiments were described
above to aid in understanding the present invention, but the
present invention shall not be construed as being limited to the
embodiments described above.
* * * * *