JP4222877B2 - Microscope observation method and microscope used therefor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microscope observation method and a microscope for using the same, and in particular, when a phase object such as a living cell can be observed over a wide range, or fine irregularities formed on a semiconductor or a glass substrate. The present invention relates to an observation method for observation and a microscope used therefor.
[0002]
[Prior art]
Living cells and the like are colorless and transparent objects, and such colorless and transparent objects are called phase objects. When observing this phase object with a microscope, special observation methods such as phase difference observation and differential interference observation are used.
[0003]
In the phase difference observation method, for example, as shown in Japanese Patent Laid-Open No. 7-225341, a ring-shaped opening is arranged at the pupil position of a condenser lens of a microscope, and a conjugate objective is formed through the ring-shaped opening and the observation object. This is an observation method in which a phase film similar to the annular opening is arranged at the pupil position of the lens, and an image contrast proportional to the phase distribution of the phase object is obtained.
[0004]
In differential interference observation, for example, as shown in Japanese Patent Laid-Open No. 8-122648, birefringent prisms are arranged at the pupil positions of the condenser lens and objective lens of the microscope, respectively, and the two orthogonally polarized components are shifted laterally. This is an observation method in which the image contrast proportional to the differential value of the phase distribution of the phase object is obtained.
[0005]
On the other hand, research is being conducted on regenerative medicine in which cells are collected from a living body, the living cells grown by culturing are returned to the collected living body, and a disease in the living body is treated.
When cells are cultured by such regenerative medicine, it is necessary to quickly observe the culture state of the cells. A petri dish or the like having a diameter of about 90 mm is usually used for culturing living cells. In order to observe the entire petri dish quickly, it is desired to observe as wide a range as possible. Therefore, in the case of research related to regenerative medicine, it is desired to confirm the culture state by observation at a lower magnification. Actually, in microscopic observation, it is 1 ×, 1.25 ×, 2 ×, 2. A low-magnification objective lens such as 5x or 4x is used.
[0006]
[Problems to be solved by the invention]
However, since a living cell is a phase object, it cannot be seen without using a phase difference observation method or differential interference observation method.
[0007]
In the phase contrast observation method and differential interference observation method, the conjugate relationship between the pupil of the condenser lens and the pupil of the objective lens is important. When a low-magnification objective lens is used, the pupil is relayed from the condenser lens to the objective lens. When this occurs, pupil aberration occurs. For this reason, it becomes difficult to maintain the conjugate relationship between the condenser lens and the pupil of the objective lens.
[0008]
For these reasons, it is difficult to perform observation using an objective lens having a lower magnification than observation using a 4 × objective lens. That is, when observing the state of cultured cells, it is difficult to observe a range wider than the field of view of a 4 × objective lens.
[0009]
The present invention has been made in view of the above situation, and provides an observation method of a microscope capable of observing a wide range and a microscope for using the same.
[0010]
[Means for Solving the Problems]
According to a first aspect of the present invention, there is provided a microscope observation method in which a phase object illuminated by light passing through a partial aperture disposed at a pupil position of an illumination optical system is deselected from a focus position of the observation optical system with respect to the phase object. By focusing, a phase difference is generated between the light transmitted or reflected by the phase object and the light diffracted by the phase object, and an image of the phase object defocused to the near point side with respect to the in-focus position And a phase object image defocused on the far point side are respectively captured and stored, and a difference image between the two stored images is calculated to form a visualized image of the phase object.
A microscope observation method according to a second aspect is characterized in that, in the first aspect, a defocus amount at the time of capturing an image of the phase object is set for each phase object.
According to a third aspect of the present invention, there is provided a microscope observation method according to the first aspect, wherein the partial aperture is eccentrically arranged in the illumination optical system.
According to a fourth aspect of the present invention, in the microscope observation method according to the third aspect, the partial opening is an annular opening.
According to a fifth aspect of the present invention, in the microscope observation method according to the first aspect, the visualized image of the phase object forms an image contrast of the phase object in a relief shape.
[0011]
A phase object observation microscope according to a sixth aspect of the present invention includes an illumination optical system having a partial aperture at a pupil position, an observation optical system for observing a phase object illuminated by the illumination optical system, and an image of the phase object. Imaging means for capturing and storing the image, and image processing means for performing image processing on the stored image of the phase object, and the phase object defocused to the near point side with respect to the focus position of the observation optical system An image and a phase object image defocused to the far point side are respectively captured and stored, and a difference image between the two stored images is calculated to form a visualized image of the phase object.
A phase object observation microscope according to a seventh aspect is characterized in that, in the sixth aspect, a defocus amount when an image of the phase object is captured is set for each phase object.
The phase object observation microscope according to claim 8 is characterized in that, in claim 6, the partial aperture is arranged eccentrically in the illumination optical system.
The phase object observation microscope according to claim 9 is characterized in that, in claim 8, the partial opening is an annular opening.
[0012]
Here, the principle of generating image contrast proportional to the phase distribution of living cells using the present invention will be described.
[0013]
FIG. 1 is a diagram for explaining the principle, and schematically shows an object point, an optical system entrance pupil, an exit pupil, and an imaging plane. As shown in this figure, when the observation object is placed at the in-focus position, the light emitted from the observation point spreads into a spherical shape as shown by the solid line and enters the entrance pupil. The light that enters the entrance pupil becomes spherical convergent light from the exit pupil and is condensed on the image forming surface to form an image. At this time, since there is no optical path (phase) difference between the light beams transmitted through the optical system, no blur occurs in the image.
[0014]
However, when the object point is moved to the position indicated by the dotted line, the light from the object point enters the entrance pupil in a spherical shape. The light that has entered the entrance pupil changes to spherical light that travels from the exit pupil toward the image plane after movement. When observed on the image plane after movement, no optical path (phase) difference occurs in the light, but when observed on the original imaging plane without moving the observation point, an optical path (phase) difference occurs in each light beam.
[0015]
In the present invention, by utilizing this, a phase difference is generated in each light beam transmitted through the optical system by arranging the observation object at a position shifted from the focus position of the optical system.
In FIG. 1, the phase difference between the optical axis of the optical system and the maximum NA ray is the largest.
[0016]
On the other hand, living cells used for culture observation have substantially the same shape. Therefore, when light is incident on cultured cells distributed in a petri dish, diffracted light is emitted in a specific direction depending on the shape of the cells. appear. When illumination light is incident on cells in the petri dish from a specific direction, transmitted light is generated in the direction of the incident light, and diffracted light is generated at a specific angle with respect to the transmitted light.
[0017]
In the present invention, the observation object is irradiated with illumination light having a specific angle by disposing the opening at the pupil position of the illumination optical system of the microscope so as to be decentered with respect to the optical axis of the illumination optical system. Since the aperture is decentered, diffracted light can be incident on the entrance pupil of the optical system with an angle with respect to the transmitted light transmitted in the direction of the incident light on the observation object. The phase difference can be increased.
[0018]
That is, in the present invention, by observing the observation object at a position deviating from the focus position of the observation optical system, the amount of deviation (defocus) from the focus position between the light transmitted through the observation object and the diffracted light is observed. A phase difference amount corresponding to the (quantity) can be generated. Further, by arranging the aperture to be decentered from the optical axis of the illumination optical system, the diffracted light can be more angled with respect to the transmitted light. That is, the phase difference between the transmitted light and the diffracted light can be further increased.
[0019]
Therefore, the amount of this phase difference functions equivalently to the phase film used in the phase difference observation method, and can provide an image contrast proportional to the phase distribution of the cultured cells, observing colorless and transparent cultured cells. I can do it.
[0020]
At this time, when the magnification of the observation optical system is low, it becomes easier to limit the angle of the diffracted light passing through the observation optical system, and the contrast of the image is improved.
When observing a phase object, the image contrast proportional to the phase distribution of the phase object is proportional to the phase amount of the observation object and the phase difference amount provided between transmitted light and diffracted light. The angle between the light diffracted by the observation object and the transmitted light varies depending on the shape of the observation object. When the angle between the diffracted light and the transmitted light changes, the amount of phase difference generated between the two light beams differs even with the same defocus amount.
[0021]
Better image contrast can be obtained by changing the defocus amount for each cell to be observed.
The sign of the phase difference of each light beam caused by defocusing changes depending on whether the observation object is shifted to the near point side or the far point side from the focus position of the observation optical system.
[0022]
An image observed by shifting an observation object such as a cultured cell from the in-focus position of the observation optical system to the near point side and an image observed by shifting the observation object to the far point side are images respectively obtained corresponding to the phase distribution of the observation object. Image contrast is reversed.
[0023]
Further, an object that absorbs light such as dust adhering to a petri dish is no longer a phase object, and even if a phase difference is given by defocusing, the image contrast does not change.
[0024]
Therefore, an image component that is not affected by the phase difference given by the defocusing can be separated by performing an inter-image calculation on the image captured by shifting to the near point side and the image captured by shifting to the far point side. In particular, by performing the difference calculation for each pixel of the two images, the image contrast of the image component corresponding to the phase distribution of the observation object can be doubled, and phase information such as dust, foreign matter, and illumination unevenness can be obtained. It is possible to eliminate unwanted image components.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
(First embodiment)
The configuration of the first exemplary embodiment of the present invention will be described with reference to FIGS.
[0026]
FIG. 2 is a schematic view of an inverted microscope, FIG. 3 is a view showing the shape of the opening, and FIG. 4 is a projection view of the opening.
As shown in FIG. 2, an inverted microscope 1 includes a light source 2, an illumination optical system 3 for guiding light emitted from the light source 2, an observation optical system 5 for forming an image of the observation object 4, and an observation. An eyepiece 6 for enlarging and visually observing the image of the object 4 and a CCD camera 8 having an image sensor 7 for capturing an image of the observed object 4 are configured. The CCD camera 8 is connected to a television monitor (not shown).
[0027]
The illumination optical system 3 includes, in order from the light source 2 side, a collector lens 10, a deflection mirror 12 for deflecting the optical axis 11 of illumination light, an interference filter 13 for making the light source 2 quasi-monochromatic, and a condenser lens 14. Has been.
[0028]
The observation optical system 5 includes, in order from the observation object 4 side, an objective lens 15 for forming an image of the observation object 4, a first relay lens 16, a deflection mirror 17 for deflecting light from the objective lens 15, a first mirror. The first relay lens 16 and the second relay lens 18 are used to form an image of the objective lens 15 (an image of the observation object 4) on the imaging surface.
[0029]
In addition, a half mirror is provided between the second relay lens 18 and the imaging plane 19 so that the visual observation by the eyepiece 6 and the observation by the CCD camera can be switched to the same or arbitrary for the image of the observation object 4. A switching mirror 20 is arranged.
[0030]
Further, an aperture unit 27 having a partial ring zone aperture 26 shown in FIG. 4 is detachably disposed at the pupil position 25 of the condenser lens 14 on which the image of the light source 2 is projected. The opening 26 itself is arranged so as to be eccentric with respect to the center of the pupil. Further, the portions other than the opening 26 of the opening unit 27 are formed from a light shielding plate. As shown in FIG. 5, the opening 26 is formed in a size that is projected so as to be substantially inscribed in the outer peripheral portion of the pupil of the objective lens 15. Further, it is desirable that the width (Ro-Ri) of the opening 26 is equal to or less than one third of the pupil radius of the objective lens 15 having a conjugate relationship. It is possible to obtain the best image contrast by appropriately setting the amount of movement according to the type of living cell to be observed.
[0031]
Next, the operation of the first embodiment will be described.
The light source 2 is projected onto the pupil position of the condenser lens 14. The light source 2 image illuminates the observation object 4 as Koehler illumination by the condenser lens 14. The light that illuminates the observation object 4 passes through the observation object 4 and enters the objective lens 15. The light incident on the objective lens 15 forms an image of the observation object 4 on the image plane 19 by the objective lens 15, the first relay lens 16, and the second relay lens 18.
[0032]
The image of the observation object 4 formed on the imaging surface 19 enters the eyepiece 6 and is imaged on the imaging surface of the imaging device 7 of the CCD camera 8 by the switching mirror 20.
On the other hand, when the aperture unit 27 is arranged at the pupil position of the condenser lens 14, the position of the aperture 26 is adjusted so that the image of the aperture 26 is substantially within the outer peripheral portion of the pupil of the objective lens 15 as shown in FIG. The object 4 is projected so as to be in contact, and the observation object 4 is obliquely illuminated from a specific direction at the same angle as the maximum NA of the objective lens 15.
[0033]
The oblique illumination light is incident on a living cell having a three-dimensional size to be cultured as the observation object 4, and is decomposed into transmitted light, refracted light, and diffracted light and emitted from the cell. In the part where the outline of the cell has a shape close to a sphere or ellipsoid, more light is refracted, and in the flat part, transmitted light and diffracted light are increased. The light refracted near the cell sphere or ellipsoid is partly larger than the NA of the objective lens 15 and is not taken into the objective lens 15.
[0034]
In the present invention, the position of the objective lens 15 is moved by a minute amount either forward or backward with respect to the observation object 4 from a position that is a normal observation focus position. As a result, a phase difference proportional to the amount of movement is generated in each light beam transmitted through the observation optical system 5, and the light beam that passes through the maximum NA of the objective lens 15 is maximized. At this time, part of the diffracted light is diffracted outside the NA of the objective lens 15 by the oblique illumination, and is not transmitted through the observation optical system 5, and a relief-like image contrast is formed as in the case of the refracted light. .
[0035]
Further, the amount of movement for moving the objective lens 15 back and forth needs to be within a range in which the formed image is not blurred, and the NA of the objective lens 15 that uses the wavelength of the light source 2 used for observation is 2 It is desirable to be less than or equal to the value divided by the power.
[0036]
The effect of the first embodiment will be described.
In the present invention, the position of the objective lens is moved by a minute amount with respect to the observation object 4 from the normal observation focus position, so that the same objective as the phase difference observation method can be used without using the phase difference objective lens. Image contrast can be obtained.
[0037]
Since it is not necessary to consider the pupil aberration between the objective lens and the condenser lens, a lower magnification objective lens can be used. In other words, phase objects such as cultured cells can be observed even with the use of 1x or 2x objective lenses that cannot be used for phase contrast observation, and a wider range of observations that could not be realized with conventional observation methods. Is possible.
[0038]
Furthermore, a relief-like image contrast in which the phase distribution is shaded can be obtained by the oblique illumination.
Moreover, visual observation and observation with a television monitor can be performed by the switching mirror.
[0039]
If the opening is removed, normal microscopic observation is possible.
(Second Embodiment)
The second embodiment will be described with reference to FIGS. 5 and 6.
[0040]
FIG. 5 is a view showing the shape of the opening, and FIG. 6 is a projection view of the opening.
In this embodiment, the aperture unit 30 shown in FIG. 5 is arranged at the pupil position of the condenser lens of the first embodiment, and the others are the same as those of the first embodiment.
[0041]
As shown in FIG. 5, the aperture unit 30 includes a rectangular aperture 31 at a position away from the center, and other portions are formed from a light-shielding light-shielding plate. The aperture 31 itself is located with respect to the center of the pupil. Are arranged in an eccentric state.
[0042]
In the second embodiment, the observation object 4 is obliquely illuminated by the rectangular opening 31 of the opening unit 30, and the opening 31 is projected as shown in FIG.
In the second embodiment, since the rectangular openings are not arranged concentrically with respect to the center of the pupil, it is possible to improve the contrast, for example, when observing elongated cells.
(Third embodiment)
The configuration of the third embodiment will be described with reference to FIG. 2, FIG. 7, and FIG.
[0043]
FIG. 7 is a view showing the shape of the opening, and FIG. 8 is a projection view of the opening.
In the third embodiment, a personal computer (not shown) for storing and calculating an image from the CCD camera 8 is connected to the CCD camera 8 of FIG. Furthermore, a television monitor (not shown) is connected to the personal computer.
[0044]
An aperture unit 40 shown in FIG. 7 is arranged at the pupil position 25 of the condenser lens 14 of the illumination optical system 3 shown in FIG. Unlike the opening 26 of the first embodiment, the opening unit 40 has a ring-shaped opening 41 that exists over substantially the entire circumference, and the others are formed of a light shielding plate. The opening 41 itself is arranged so as to be eccentric with respect to the center of the pupil. Further, as shown in FIG. 7, the opening 41 is substantially inscribed in the outer periphery of the pupil of the objective lens 15 so that annular illumination for illuminating the observation object 4 at an angle close to the maximum NA of the objective lens 15 can be performed. It is formed in various dimensions.
[0045]
The operation of the third embodiment will be described.
Unlike the first embodiment, the position of the opening 41 is adjusted so that the position of the opening 41 is adjusted and the light from the light source 2 that has passed through the opening 41 is in the optical axis with respect to the observation object 4. It is illuminated symmetrically.
[0046]
By switching the switching mirror 20 to the CCD camera 8, the image of the observation object 4 is displayed on the television monitor via the CCD camera 8.
The position of the objective lens 15 is slightly moved to the observation object 4 side (near point side) to capture an image and store it as a near point image in a personal computer. Next, the objective lens is moved to the opposite side (far point side) across the focal point, and the image is picked up and stored in the personal computer as a far point image.
[0047]
The personal computer calculates (near point image)-(far point image) or (far point image)-(near point image) for each pixel corresponding to each of the two stored images.
[0048]
The effect of the third embodiment will be described.
In the case of the third embodiment, unlike the first embodiment, since it is illuminated symmetrically with respect to the optical axis, the image does not have the same directionality as the phase difference observation method, rather than a relief image contrast. Can be obtained.
[0049]
By calculating the (near point image)-(far point image) or (far point image)-(near point image) for each corresponding pixel of the near point and far point images stored in the personal computer, The image contrast proportional to the phase distribution of the observation object 4 is enhanced twice. Further, the components other than the phase distribution of the observation object 4 have an image contrast of substantially 0, and it is possible to emphasize an image component proportional to the phase distribution originally desired to be observed.
(Fourth embodiment)
The configuration of the fourth embodiment will be described with reference to FIG.
[0050]
FIG. 9 is a view showing the turret 50.
In this embodiment, a turret 50 as shown in FIG. 9 is arranged at the pupil position 25 of the condenser lens 14. The turret 50 has a plurality of opening mounting holes 52 concentrically with respect to the rotating shaft 51, and various types of openings can be mounted. The turret 50 is configured to be rotatable with respect to the rotation axis so that the center point of each of the plurality of aperture mounting holes substantially coincides with the optical axis of the illumination optical system 3, and the objective lens used and the observation method are used. By rotating the turret 50, the opening mounting hole can be selected as appropriate.
[0051]
In the plurality of opening mounting holes, for example, openings having a shape shown in FIGS. 3, 5 and 6 and having a size suitable for the magnification of the objective lens are mounted.
In the fourth embodiment, since the shape and size of the opening can be easily changed in accordance with the magnification conversion of the objective lens by the rotation of the turret, good observation can be performed.
(Fifth embodiment)
In the first to fourth embodiments, examples applied to observation of living cells have been shown. The fifth embodiment is applied to the observation of an object that can be regarded as a phase object, that is, the case of observing fine irregularities formed on a semiconductor or a glass substrate. This can be considered that the minute unevenness on the surface is a phase component created by the minute step, and it is effective to use the present invention.
[0052]
Observation of fine irregularities formed on a semiconductor surface or a glass substrate uses an episcopic microscope shown in FIG. 10, and will be described using an episcopic microscope.
The epi-illumination microscope 60 includes a light source 61, an illumination optical system 63 for guiding the light emitted from the light source 61 to the observation object 62, an observation optical system 64 for forming an image of the observation object 62, and the observation object 62. A CCD camera 66 having an image pickup element 65 for picking up an image. The CCD camera 66 is connected to a television monitor (not shown).
[0053]
The illumination optical system 63 includes, in order from the light source 61 side, a collector lens 67, an illumination relay lens 68, a half mirror 70 for deflecting the optical axis 69 of illumination light, and an objective lens 71 that also constitutes an observation optical system 64. Yes.
[0054]
An opening unit 72 is detachably disposed at a position where the image of the light source 61 is projected. The shape of the opening attached to the opening unit 72 is as shown in FIGS. 3, 5 and 6, and the size of the opening is determined according to the size of the pupil of the objective lens.
[0055]
The observation optical system 64 includes, in order from the observation object 62 side, an objective lens 71 for forming an image of the observation object 62 and an imaging lens 72 for forming an image of the objective lens 71.
[0056]
The operation of the fifth embodiment will be described.
The light emitted from the light source 61 forms an image of the light source 61 in the illumination optical system 63 by the collector lens 67. The light source 61 image is deflected by the illumination relay lens 68 by the half mirror 70 and then relayed to the pupil position of the objective lens 71 to form the light source 61 image again. Then, the relayed light source 61 image Koehler-illuminates the observation object 62 by the objective lens 71. At this time, the objective lens 71 also functions as a condenser lens.
[0057]
The light that illuminates the observation object 62 is reflected by the observation object 62 and enters the objective lens 71. The light incident on the objective lens 71 passes through the half mirror 70 and forms an observation object 62 image on the imaging surface by the imaging lens 72. The CCD camera 66 is placed on the image plane, and an image of the observation object 62 is projected on the television monitor via the CCD camera 66 for observation.
[0058]
In the present embodiment, the opening unit 72 is arranged at a position where the image of the light source 61 is formed by the collector lens 67. Further, the observation object 62 is moved back and forth by a small amount from the normal focus position, and the image contrast of the observation object 4 is adjusted.
[0059]
The effect of the fifth embodiment will be described.
In the fifth embodiment, a semiconductor or a glass substrate can be observed as an observation object. That is, minute unevenness on the surface of a semiconductor or glass substrate can be observed as a phase component created by a minute step.
[0060]
Further, if the opening having the shape shown in FIG. 3 or FIG. 5 is used, an image contrast with a relief feeling can be obtained.
Further, if the opening having the shape shown in FIG. 6 is used, an image contrast having no directionality can be obtained.
[0061]
Further, in order to enhance contrast, it is effective to take an image at a position shifted by two places before and after the in-focus position and perform a difference calculation thereof.
In the embodiments described so far, the annular opening is arranged so as to be inscribed in the outer peripheral portion of the objective lens pupil. However, the circular pinhole opening may be arranged in the center of the pupil in the same manner. The same image contrast as the phase difference observation method can be obtained. In this case, it is desirable to adjust the position of the pinhole so that it is in the vicinity of the approximate center of the pupil of the objective lens having a conjugate relationship, and an image contrast having no directionality can be obtained.
[0062]
【The invention's effect】
According to the present invention can be favorably to observe phase object range have wide.
[Brief description of the drawings]
FIG. 1 is a diagram for explaining the principle. FIG. 2 is a schematic diagram of an inverted microscope. FIG. 3 is a diagram showing the shape of an opening according to the first embodiment. FIG. 4 is a projection view of the opening. The figure which shows the shape of the opening of the form of [FIG. 6] The projection figure of an opening [FIG. 7] The figure which shows the shape of the opening of 3rd Embodiment [FIG. 8] The projection figure of an opening [FIG. 9] The figure which shows a turret [Figure 10] Schematic diagram of epi-illumination microscope [Explanation of symbols]
14 ... Condenser lens 26 ... Opening 27 ... Opening unit 30 ... Opening unit 31 ... Opening 40 ... Opening unit 41 ... Opening 50 ... Turret

Claims (9)

The phase object illuminated by the light passing through the partial aperture arranged at the pupil position of the illumination optical system is defocused from the in-focus position of the observation optical system with respect to the phase object, thereby transmitting or reflecting the phase object. A phase difference is generated between the light and the light diffracted by the phase object, and an image of the phase object defocused to the near point side with respect to the in-focus position and an image of the phase object defocused to the far point side A microscope observation method characterized in that a visualized image of the phase object is formed by imaging and storing each of them and performing a difference calculation between the two stored images . The microscope observation method according to claim 1, wherein a defocus amount when capturing an image of the phase object is set for each phase object. The microscope observation method according to claim 1, wherein the partial aperture is arranged eccentrically in the illumination optical system. The microscope observation method according to claim 3, wherein the partial opening is an annular opening. The microscope observation method according to claim 1, wherein the visualized image of the phase object forms an image contrast of the phase object in a relief shape. An illumination optical system with a partial aperture at the pupil position;
  An observation optical system for observing the phase object illuminated by the illumination optical system;
  Imaging means for capturing and storing an image of the phase object;
  Image processing means for performing image processing on the stored image of the phase object;
With
  The image of the phase object defocused to the near point side and the image of the phase object defocused to the far point side with respect to the in-focus position of the observation optical system are respectively captured and stored, and the stored 2 A phase object observation microscope characterized by forming a visualized image of the phase object by performing an image difference calculation. The phase object observation microscope according to claim 6, wherein a defocus amount for capturing an image of the phase object is set for each phase object. The phase object observation microscope according to claim 6, wherein the partial aperture is arranged eccentrically in the illumination optical system. The phase object observation microscope according to claim 8, wherein the partial opening is an annular opening.

Images