JPH1020199A - Compound microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To observe a sample in a culture container by means of an optical microscope and to facilitate probe scanning by arranging a probe microscope unit and an illumination optical system on an opposite side to an objective lens by interposing the sample in between and arranging the illumination optical system so that it may be inclined to the optical axis of the objective lens. SOLUTION: The probe microscope is arranged coaxially with an observation optical system by interposing a sample surface 6. After illuminating light from a light source 1 is spread by a corrector lens 2, it is condensed on the sample surface 6 by a condenser lens 4 through an aperture diaphragm 3. The inclination of the eccentric illumination optical system to the optical axis 5 is varied, so that the position of the transmitted light passing through the objective lens 7 is changed. The range of the transmitted light passing through the lens 7 is varied in the diaphragm 3 arranged in the eccentric illumination optical system. The transmitted light forms the image of the sample at an image position 32 through the lens 7. An angle formed by the optical axis 5 of the lens 7 with the optical axis of the eccentric optical system is θ=28 deg..

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a scanning microscope, and more particularly to a scanning tunneling microscope (STM), an atomic force microscope (AFM), and a near-field scanning optical microscope (NS).
OM) and other devices that measure the surface shape of a sample by using the interaction between the sample and the probe, such as a high-magnification observation of a living biological specimen with an optical microscope, and a complex function that can perform various operations on the specimen. The present invention relates to a microscope having:

[0002]

2. Description of the Related Art In a scanning probe microscope, a probe (probe) is brought close to a sample, and physical quantities such as current and force generated therebetween are measured, and the state of the surface of the sample is imaged based on this. It is a device to do. Specimen imaging is performed by scanning the probe, but the interval between the specimen and the probe is extremely narrow and high-speed scanning cannot be performed. Therefore, it takes a lot of time to repeatedly scan to determine the observation range. Occurs. Therefore, a method of setting an approximate scanning range in combination with an optical microscope is disclosed in JP-A-1-801603 and JP-A-2-16403. JP-A-1-801603
The publication discloses a technique in which a probe of a scanning tunneling microscope and an objective lens of an optical microscope match each other, so that the field of view of an observation surface of the scanning tunneling microscope can be observed as an image of the optical microscope. JP-A-2-
No. 16403 uses an optical microscope to set a range for observation by a scanning tunneling microscope, and an objective lens and a probe are used to improve the positioning accuracy of the probe within the observation field of the optical microscope. This is an integrated technology.

[0003]

In recent years, there has been an increasing demand for using a scanning probe microscope for observing and measuring, at a high resolution, a living biological specimen contained in a culture vessel or the like. Living biological specimens are generally colorless and transparent,
Special observation such as phase contrast observation with an optical microscope is required for setting an observation range with a scanning probe microscope. In addition, since the observation is performed in the culture vessel, the observation is performed by transmission observation using an inverted microscope by bringing the objective lens close to the bottom surface of the culture vessel.

[0004] However, Japanese Patent Application Laid-Open Nos.
In Japanese Patent Application Laid-Open No. 2-16403, a probe and an objective lens are arranged on the same side with respect to a specimen because the observation of a reflection specimen such as a metal is assumed. Therefore, even if the probe is used as it is in an inverted microscope, the probe is blocked by the bottom surface of the culture vessel and cannot be observed by the probe. In addition, illumination for observation with an optical microscope is also epi-illumination, and in this configuration, special observation such as phase difference observation cannot be performed on a sample placed in a culture vessel.

The present invention has been made in view of such a problem, and an object of the present invention is to provide a compound microscope that enables a specimen in a culture vessel to be observed with an optical microscope and that can easily perform probe scanning. And

[0006]

In order to achieve the above object, a compound microscope according to the present invention comprises: an optical microscope having an illumination optical system and an observation optical system having an objective lens;
In a compound microscope having a probe microscope unit arranged on the optical axis of the objective lens, the probe microscope unit and the illumination optical system are arranged on the opposite side of the objective lens across a sample, and the illumination optical system is It is characterized by being arranged obliquely with respect to the optical axis of the objective lens.

Here, the inclination of the optical system preferably satisfies the following condition when the angle between the optical axis of the illumination optical system and the optical axis of the objective lens is θ. sin | θ | ≧ 0.4 Further, when the pupil modulation optical element is arranged at the pupil of the objective lens, in the vicinity thereof, or at a position substantially conjugate with them, the illumination light passing through the sample is provided on the pupil modulation optical element. It is effective if the inclination of the illumination optical system is adjusted so as to pass through the modulation area and the illumination light satisfies the following conditions.

NA ≧ (NA ₁ −NA ₂ ) / 2 where NA ₁ is the numerical aperture corresponding to the position farthest from the optical axis in the modulation area of the pupil modulation optical element, and NA ₂ is the pupil modulation optical element. Is the numerical aperture corresponding to the position closest to the optical axis in the modulation area, and NA is the numerical aperture of the illumination optical system. Further, a second illumination optical system different from the above illumination optical system is arranged on the optical axis of the objective lens so as to illuminate from behind the probe microscope unit. At this time, it is preferable that the second pupil modulation optical element is disposed in the observation optical system at the pupil of the objective lens, in the vicinity thereof, or at a position substantially conjugate with them. The pupil modulation optical element and the second pupil modulation optical element can be integrally molded and used.

[0009] In these compound microscopes, it is preferable that the illumination optical system has a variable stop. further,
The observation optical system may include an imaging device, and an image signal of the imaging device may be displayed as an image through an image processing device. Note that the output of the probe microscope unit can be input to the image processing apparatus.

Hereinafter, the reason and operation of the above configuration in the present invention, and a more preferable configuration will be described. The basic concept of the present invention will be described with reference to FIG. FIG. 2 is a diagram showing a system diagram of the compound microscope of the present invention.
0, an image processing device 21, and an image display device 22. The compound microscope is a combination of an inverted optical microscope and a probe microscope.

The optical microscope includes an inverted microscope main body 204, an illumination optical system 23, a stage 209 for holding a culture vessel 208 containing a sample, an objective lens 210 disposed below the stage 209 and for observing from the bottom side of the culture vessel. And an eyepiece 211 and an imaging device 212. The illumination optical system 23 is a coaxial illumination optical system 2 conventionally used.
05 and an eccentric illumination optical system 206 which is arranged obliquely with respect to the optical axis of the objective lens. Eccentric illumination optical system 2
The illumination angle of 06 can be adjusted by the angle adjusting member 207.

On the other hand, the probe microscope unit 25 includes a probe 201 disposed above the stage 209 of the inverted microscope 204, and a probe holder 2 for holding the probe 201.
02, a probe scanning unit 203 that scans the probe, and is integrated with the inverted microscope 204. Since the probe holding unit 202 is disposed right above the sample, the probe 201 is stable, and good sample information can be obtained.

Signals from the imaging device 212 and the probe microscope unit 25 are sent to the image processing device 21 and subjected to appropriate processing, and then the image of the sample is displayed on the image display device 22. In such a configuration, observation of the sample is performed as follows. The specimen is placed in a culture vessel 208 placed on a stage 209. When observing an optical image, the illumination optical system 23 illuminates the culture vessel 208 from above, and the objective lens 210 passes through the eyepiece from the bottom. Observe by 211. The eccentric illumination optical system 206
The angle at which the specimen is illuminated can be adjusted by the angle adjusting member 207 according to the magnification and type of the objective lens. Further, an aperture stop is arranged inside the eccentric illumination optical system 206 so that the range of illumination light can be adjusted.

The optical path can be switched inside the inverted microscope 204, and light from the sample can be guided to the image pickup device 212 to photograph the sample. on the other hand,
The observation by the STM image or the AFM image is performed by moving the probe 201 from the upper part of the culture vessel 208 to the sample and moving the probe 201 closer to the specimen.
Image information is obtained by performing Y scanning. An output signal from the probe microscope unit 25 is input to the image processing device 21, subjected to appropriate image processing, and displayed on the image display device 22. The image processing device 21 includes the imaging device 2
Twelve outputs can also be connected.

With such a configuration, even if the sample is a colorless and transparent sample in the culture solution, contrast is generated in the observation image by the eccentric illumination, and observation or photographing of an optical image becomes possible. Further, since the probe 201 is disposed approximately on the optical axis of the objective lens 210, both the sample and the probe can be viewed at the same time, and the positioning of the probe can be easily performed.

Further, the provision of the coaxial illumination optical system 205 enables illumination that substantially satisfies the numerical aperture of the objective lens. Therefore, when observing a sample that is not colorless and transparent, it is possible to observe with an imaging performance close to the performance originally possessed by the objective lens. Next, the state of the decentered illumination optical system and light rays in the objective lens will be described. In microscope observation, the observation magnification is changed by exchanging the objective lens, but the numerical aperture of the objective lens differs depending on the type and magnification of the objective lens.
Therefore, in the illumination by the decentered illumination optical system as in the present invention, when the objective lens is replaced, the illumination area of the illumination light is adjusted so that the illumination light passing through the sample passes through the opening of the objective lens. You have to do it. In a phase contrast objective lens used for phase contrast observation of a colorless and transparent sample, such adjustment is particularly necessary because the position and area of the phase film on the phase plate are determined.

There is a method of illuminating obliquely as a method of giving contrast to an image of a colorless and transparent sample. In this case, it is desirable to illuminate the image at a large oblique angle. Here, if the above illumination is performed by coaxial illumination,
As shown in FIG. 1A, a light beam that can be illuminated according to the numerical aperture of the objective lens 7 is secured in advance, and a part of the light beam is extracted by the aperture stop 3 '. The illumination aperture that has passed through the aperture stop 3 ′ is condensed on the sample surface 6 by the condenser optical system 4. The adjustment of the illumination range is performed by the aperture stop 3 '.
The opening is moved closer to or farther from the optical axis.

However, when the probe microscope unit exists on the optical axis of the objective lens as in the present invention, such a configuration is practical because the illumination optical system and the probe microscope unit structurally interfere with each other. Is impossible. Even if the probe microscope unit does not exist, only a part of the illuminating light beam is used, so that the efficiency of using the illuminating light is inferior. There is a problem in that it must be large.

On the other hand, in the present invention, as shown in FIG. 1B, an eccentric illumination optical system is employed for the illumination system. In FIG. 1B, the decentered illumination optical system includes a light source 1, a collector lens system 2, an aperture stop 3, and a condenser lens system 4. Reference numeral 7 denotes an objective lens. Illumination light from the light source 1 is irradiated onto the sample surface 6 by the collector lens system 2 and the condenser lens system 4. The transmitted light that has passed through the sample enters the objective lens 7 and forms an image of the sample behind.

FIG. 1B illuminates a region having a large numerical aperture with respect to the numerical aperture of the objective lens 7 so that the transmitted light of the sample passes. As a result of tilting the decentered illumination optical system with respect to the optical axis 5 of the objective lens 7 at an angle θ, a numerical aperture NA _max corresponding to the outermost ray of the transmitted light from the sample surface 6 passing through the objective lens 7 There are matches the numerical aperture NA _a of the objective lens 7. As described above, the position of the light beam passing through the objective lens can be adjusted by changing the inclination angle θ in the decentered illumination optical system. Therefore, even when a phase difference objective lens is used, illumination matching the position of the phase film can be performed, so that phase difference observation can be performed without any problem. Also, at the time of observation with a high-magnification objective lens, illumination for observation using an area with a large numerical aperture can be performed without increasing the numerical aperture of the illumination optical system.

Here, it is desirable that the absolute value of the inclination angle θ of the decentered illumination optical system satisfies the following condition. sin | θ | ≧ 0.4 (1) Conditional expression (1) is a condition relating to illumination during high-magnification observation.
If the conditional expression is not satisfied, the transmitted light from the sample does not pass through a required area in the objective lens, so that the contrast and the resolution are reduced.

Next, the illumination range will be described. The illumination light flux emitted from the eccentric illumination optical system having a numerical aperture NA is 2NA
Illuminate the specimen with a width of. The transmitted light from the sample surface 6 is incident on the objective lens. Here, assuming that the numerical aperture corresponding to the innermost ray of the transmitted light passing through the objective lens is NA _min , the light flux passing through the objective lens is Range is NA _max and N
Between A _min . Since the adjustment of the width of the illumination light beam is performed by changing the size of the aperture stop arranged in the decentered illumination optical system, the aperture stop serves to adjust the range of the light beam passing through the objective lens. Therefore, even when the phase difference objective lens is used, the illumination according to the range of the phase film can be performed, so that the phase difference observation can be performed without any problem.

Further, let the numerical aperture of the illumination optical system be NA,
In the phase plate disposed at or near the pupil position in the objective lens, the numerical aperture corresponding to the light beam passing through the position farthest from the optical axis in the region of the phase film on the phase plate is defined as the maximum numerical aperture NA ₁ , Assuming that the numerical aperture corresponding to the light beam passing through the position closest to the axis is the minimum numerical aperture NA ₂ , it is desirable to satisfy the following conditions.

NA ≧ (NA ₁ −NA ₂ ) / 2 (2) That is, generally, as the numerical aperture of the illumination light decreases, the coherence increases and the depth of focus increases. An image of a certain dust or the like becomes conspicuous and overlaps with the phase difference image of the sample, obstructing observation. A similar phenomenon occurs in the decentered illumination optical system, and the phase difference image becomes difficult to see. The conditional expression (2) takes this point into consideration. If the numerical aperture of the illumination light does not satisfy the conditional expression, the above-described problem occurs, which hinders observation.

The second illumination optical system, which is arranged on the optical axis of the objective lens so as to be illuminated from behind the probe microscope unit and is arranged coaxially with the optical axis of the observation optical system, has an illumination light having a numerical aperture. In order to illuminate the low range, the light beam serves to increase the light flux satisfying the numerical aperture of the objective lens. still,
Other features of the present invention will become apparent from the following examples.

[0026]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the compound microscope of the present invention will be described. These embodiments are examples applied to the case where a phase difference objective lens is used.

[0027]

Embodiment 1 FIG. 3 shows a first embodiment of the present invention. The present embodiment is a state of decentered illumination when a phase difference objective lens is used. The same components as those in FIG. 1 are denoted by the same reference numerals, and descriptions of the functions and the like are omitted. In FIG.
The observation optical system has an objective lens 7 and an imaging lens system 30. Note that a phase plate 31 is arranged at or near the pupil position of the objective lens 7. 32 is an image position.
The decentered illumination optical system has an angle θ with respect to the optical axis 5 of the observation optical system.
It is arranged at an angle. When a relay optical system is located behind the image position 32, the phase plate 31 may be arranged at a position conjugate with the pupil position of the objective lens in the relay optical system or in the vicinity thereof.

On the other hand, the probe microscope is arranged coaxially with the observation optical system with the sample surface 6 interposed therebetween. 35 is a probe, and 34 is a probe holding unit. Illumination light from the light source 1 is spread by the collector lens 2, passes through the aperture stop 3, and is focused on the sample surface 6 by the condenser lens 4. The decentered illumination optical system has a mechanism (not shown) that can change the inclination with respect to the optical axis 5, and can change the position of transmitted light passing through the objective lens 7. The aperture stop 3 arranged in the decentered illumination optical system is a variable stop having a structure similar to that of a camera, for example, and makes the range of transmitted light passing through the objective lens 7 variable. And sample surface 6
Transmitted through the objective lens 7 passes through the imaging lens system 3
0 forms an image of the specimen at image position 32.

In this embodiment, the optical axis 5 of the objective lens 7
And the optical axis of the eccentric illumination optical system are θ = 28 °, and the numerical aperture NA of the eccentric illumination optical system is 0.08. The numerical apertures NA1 and NA2 determined by the position of the phase film 33 on the phase plate 31 in the objective lens 7 are given by NA1 =
0.5, NA2 = 0.4. FIG. 4 shows an arrangement example of the phase film 41 of the phase plate 40. Aperture stop 3 for eccentric illumination optical system
Is a circular diaphragm, so that the phase film 41 is also circular.
The sample is illuminated with a light beam having a numerical aperture of 0.08, and the direct light of the transmitted light that has passed through the sample passes through the inside of the phase film 41.

By the way, since the fixed position of the objective lens with respect to the revolver and the position of the phase film in the objective lens vary, if the arrangement shown in FIG. Not necessarily. Therefore,
The illumination position must be moved in accordance with the position of the phase film, or the objective lens must have an adjustment mechanism for adjusting the phase film to the illumination position.

FIG. 5 shows an arrangement example in which the phase films 51 are arranged concentrically in consideration of this point, and an extra adjustment mechanism for adjusting the position in the rotation direction becomes unnecessary. The solid circle 52 inside the phase plate 50 in FIG. 5 is the direct light from the sample passing through this area. Here, as shown by the circle of the broken line 53, when the luminous flux of the illumination light is increased, the depth of focus becomes shallower, and the effect of making dust and the like in the optical system inconspicuous can be obtained. Since the ratio also increases, the contrast of the image decreases, which is a problem.

Therefore, when the aperture stop 3 of the decentered illumination optical system is a rectangular stop 60 as shown in FIG. 6, the rectangular aperture 61 is projected on the pupil of the objective lens. The projected shape is such that the long side substantially contacts the inner and outer diameters of the orbicular zone with respect to the orbicular phase film 71 on the phase plate 70 as shown by the hatched portion 72 in FIG. Therefore, since the direct light passes through this range on the phase film, the luminous flux of the illuminating light can be increased without increasing the ratio of the direct light that leaves the phase film.

FIG. 8 shows another method which is an alternative to the limiting method using the aperture stop 3 in FIG. Both sides of a normal lens are cut into a shape resembling an ellipse, and this lens 80 is used for a condenser lens in an eccentric illumination optical system. Since the outer diameter is limited, the numerical aperture in the cut direction (x direction) is 0.05, and the numerical aperture in the uncut direction (y direction) is 0.08. It should be noted that since the range of transmitted light passing through the objective lens 7 cannot be varied by the limiting method based on the outer diameter of the lens as shown in FIG.

[0034]

Embodiment 2 FIG. 9 shows a second embodiment of the present invention. The same components as those in FIG. 3 are denoted by the same reference numerals, and description thereof will be omitted. This embodiment has a coaxial illumination optical system in addition to the decentered illumination optical system. In this embodiment, the probe holding member 90 has a hollowed-out central portion, and coaxial illumination is performed using this space.

The coaxial illumination optical system has substantially the same configuration as the decentered illumination optical system, and includes a light source 91, a collector lens system 92, an aperture stop 93, a mirror 94, and a condenser lens system 95. The numerical aperture of the coaxial illumination optical system is 0.1. Since the probe holding member 90 is moved by the scanning of the probe, there is no optical element related to illumination at the center of the probe holding member 90, and only illumination light passes therethrough. The coaxial illumination optical system is attached to the optical microscope above the probe holding member 90. There is no problem if the optical elements that constitute the coaxial illumination optical system are arranged in a line on the optical axis,
In the case where the height is restricted, the optical path may be bent by the mirror 94 in a direction that does not contact the eccentric illumination optical system as shown in FIG. In FIG. 9, the mirror 94 is arranged between the aperture stop 93 and the condenser lens system 95 to bend the optical path, but the mirror 94 may be arranged at another position.

FIG. 10 shows ordinary coaxial illumination (A) and the present embodiment (B) for illumination when observing with a high-magnification objective lens.
Are compared. In ordinary coaxial illumination, illumination is performed so as to satisfy the numerical aperture NA _ob of the objective lens 7. However, in the present invention, eccentric illumination is basically performed avoiding the probe microscope unit. It can be illuminated only by the light flux a that satisfies a part of the number. However, in this embodiment, since the light flux b of the coaxial illumination optical system is added in addition to the decentered illumination optical system, illumination is performed with a numerical aperture close to that of ordinary coaxial illumination. For this reason, the coherence of the illumination light can be reduced, and a phenomenon in which an image of dust or the like other than the focal plane appears to overlap with the observation image can be suppressed. Further, even when a non-colorless and transparent sample is observed in a bright field, the illumination is performed with substantially the same numerical aperture as that of ordinary coaxial illumination.

In this embodiment, the first phase difference observation is performed.
When the same phase plate 33 as in the embodiment is used, there is no phase film at a position where the direct light from the specimen by the coaxial illumination passes. For this reason, a phase shift occurs between the direct light from the specimen due to the eccentric illumination and the direct light from the specimen due to the coaxial illumination, so that the contrast of the phase difference image is reduced. Therefore, the phase plate 110 used in this embodiment is, as shown in FIG.
In addition to the phase film 111 for eccentric illumination, a phase film 112 is arranged at the center of the phase plate 110 in accordance with the numerical aperture of coaxial illumination. In this way, the phase shift of the direct light from the sample due to the eccentric illumination and the coaxial illumination is eliminated, and the contrast of the phase difference image can be increased while keeping the coherence low.

Since a part of the diffracted light from the specimen due to the eccentric illumination passes through the phase film 112, a phase shift occurs between the diffracted light and the direct light. It is not a problem that reduces the contrast. Further, the phase films 111 and 112 may be provided on different phase plates. In this way, when the coaxial illumination is eliminated and only the eccentric illumination is used, the phase plate provided with the phase film 112 can be taken out of the optical path, so that the phase difference observation with the eccentric illumination can be performed well.

Although the embodiments have been described above, the aperture stop and the phase plate may be changed as follows. For example, as for the aperture stop, the aperture stop 3 of the decentered illumination optical system may be used alone or in combination with a plurality of stops of various sizes and shapes such as a rectangle and a circle. FIG.
A combines a circular stop 120 and a rectangular stop 121,
This is an example in which an elliptical opening is formed. These two stops 120 and 121 may be arranged close to each other on the optical axis of the aperture stop position 3 or 93 of the illumination optical system as shown in FIG. 12B. In order to change the size of the stop, a stop having a variable aperture mechanism may be used, or a stop having a different size or shape of the aperture may be replaced.

In each embodiment, the phase difference observation using the phase difference objective lens has been described. However, instead of the phase plate, a filter 130 having a plurality of regions having different transmittance distributions as shown in FIG. May be arranged at or near the pupil position. In FIG. 13A, the filter 130 has a circular area 131 that transmits light,
Reference numeral 31 denotes a semicircular portion whose transmittance changes stepwise as shown in FIG. 13B. Accordingly, as shown in FIG. 13C, the light that has passed through the sample passes through portions of the circular region 131 having different transmittances due to the phase gradient of the sample, so that the phase gradient is converted into a change in intensity. By arranging the modulation optical elements such as the phase plate and the filter as described above at or near the pupil position of the objective lens and at a position substantially conjugate with them, it is possible to observe a colorless and transparent sample. it can.

[0041]

Since the present invention is configured as described above, it has the following effects. By arranging the illumination optical system eccentrically with respect to the optical axis of the objective lens, it is possible to secure the illumination numerical aperture for high-magnification observation even if the numerical aperture of the illumination optical system is not so large. Further, by providing a variable aperture, the range and position of the partial light beam are adjusted according to the objective lens, and even if the sample is colorless and transparent, a contrast is generated in the observation image such that its location can be confirmed. And the specimen can be aligned with the high-magnification objective lens.

Further, since the numerical aperture of the illumination optical system is small, the optical system becomes compact, and physical interference with the probe microscope does not occur. Also, in modulation contrast observation in which a filter whose transmittance distribution changes at the pupil position of the objective lens, illumination light can be adjusted in accordance with the filter, so that good observation can be performed. Further, by arranging a plurality of aperture stops, when a phase difference objective lens is used, the illumination light is applied so that the light passes directly through a region of the phase film or the absorption film arranged substantially at the pupil position of the phase difference objective lens. Since the adjustment can be performed, the phase contrast image of the sample can be observed with good contrast.

Further, since the coaxial illumination optical system can be used together by making the probe holding portion hollow, the contrast can be further improved as compared with the case where only the eccentric illumination optical system is used, and the depth of focus can be reduced to reduce the optical system. It is possible to make the dust attached to the surface inconspicuous.

[Brief description of the drawings]

FIG. 1 is a diagram showing a relationship between numerical apertures in an optical system according to the present invention.

FIG. 2 is a system diagram of the compound microscope of the present invention.

FIG. 3 is a diagram of a main optical system of the compound microscope according to the first embodiment of the present invention.

FIG. 4 is a diagram showing a shape of a phase film and a transmission range of directly transmitted light in the first embodiment of the present invention.

FIG. 5 is a diagram showing another shape of the phase film and a transmission range of directly transmitted light in the first embodiment of the present invention.

FIG. 6 is a diagram showing the shape of the stop of the decentered optical system according to the first embodiment of the present invention.

FIG. 7 is a diagram illustrating a transmission range of directly transmitted light corresponding to the stop in FIG. 6 according to the first embodiment of the present invention.

FIG. 8 is a diagram illustrating a shape of a condenser lens of the decentered optical system according to the first example of the present invention.

FIG. 9 is a diagram of a main optical system of a compound microscope according to a second embodiment of the present invention.

FIG. 10 is a diagram comparing a numerical aperture in a second embodiment of the present invention with a numerical aperture in normal illumination.

FIG. 11 is a view showing a shape of a phase film in a second embodiment of the present invention.

FIG. 12 is a diagram illustrating a state of a diaphragm when a plurality of diaphragms are used in the present invention.

FIG. 13 is a diagram showing a pupil modulation optical element when performing pupil modulation by an excessive rate change in the present invention.

[Explanation of symbols]

1, 91 Light source 2, 92 Collector lens system 3, 3 ', 60, 93 Aperture stop 4, 95 Condenser lens system 5 Optical axis 6 Sample surface 7 Objective lens 120 Circular stop 121 Rectangular stop 122 Elliptical aperture stop 130 Filter 131 Circular area having step-like transmittance distribution 20 Compound microscope 21 Image processing device 22 Image display device 23 Illumination optical system 24 Observation optical system 25 Probe microscope unit 201, 35 Probe 202, 34, 90 Probe holding unit 203 Probe scanning unit 204 Inverted microscope 205 Coaxial illumination optical system 206 Eccentric illumination optical system 207 Rotating member 208 Culture vessel 209 Stage 210 Objective lens 211 Eyepiece 212 Imaging device 30 Imaging lens 31, 40, 50, 70, 110 Phase plate 32 Image position 33, 41 , 51, 7 , 111, 111, 112
Phase film 52, 53, 72 Passing range of transmitted light, 61 Aperture shape 80 Condenser lens 94 Mirror

Claims (10)

[Claims] 1. A compound microscope comprising: an optical microscope including an illumination optical system and an observation optical system having an objective lens; and a probe microscope unit disposed on an optical axis of the objective lens, wherein the probe microscope unit and A compound microscope, wherein the illumination optical system is arranged on a side opposite to the objective lens with respect to a sample, and the illumination optical system is arranged to be inclined with respect to an optical axis of the objective lens. 2. The compound microscope according to claim 1, wherein the following conditional expression is satisfied. sin | θ | ≧ 0.4 where θ is the angle between the optical axis of the illumination optical system and the optical axis of the objective lens. 3. The pupil of the objective lens, wherein the observation optical system includes:
A pupil modulation optical element is provided in the vicinity thereof or at a position substantially conjugate with the pupil modulation optical element, and the illumination optical system is arranged at an angle at which illumination light passing through the sample passes through a modulation region of the pupil modulation optical element. The composite microscope according to claim 1, wherein 4. The compound microscope according to claim 3, wherein the following conditional expression is satisfied. NA ≧ (NA ₁ −NA ₂ ) / 2 where NA ₁ is a numerical aperture corresponding to a position farthest from the optical axis in the modulation area of the pupil modulation optical element, and NA ₂ is a modulation area of the pupil modulation optical element. Is the numerical aperture corresponding to the position closest to the optical axis, and NA is the numerical aperture of the illumination optical system. 5. A second illumination optical system different from the illumination optical system, wherein the second illumination optical system is arranged on the optical axis of the objective lens so as to illuminate from behind the probe microscope unit. 3. The method according to claim 1, wherein
2. The compound microscope according to item 1. 6. A second illumination optical system different from the illumination optical system, wherein the second illumination optical system is arranged on the optical axis of the objective lens such that the second illumination optical system illuminates from behind the probe microscope unit. The compound microscope according to claim 3, wherein the observation optical system includes a second pupil modulation optical element at a pupil of the objective lens, in the vicinity thereof, or at a position substantially conjugate with the pupil. 7. The compound microscope according to claim 6, wherein the pupil modulation optical element and the second pupil modulation optical element are integrally formed. 8. The compound microscope according to claim 1, wherein the illumination optical system includes an aperture limiting unit. 9. An image pickup apparatus for picking up an image formed by the optical microscope, an image processing apparatus for processing a captured image, and an image display device for displaying an output of the image processing apparatus. The composite microscope according to claim 1. 10. The compound microscope according to claim 9, wherein an image signal from the probe microscope unit is connected to the image processing device.