JP2006220994A - Observation system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simultaneously perform pupil modulation observation such as phase difference observation, and fluorescence observation. <P>SOLUTION: The observation system 1 includes; an exciting light source 5 for emitting exciting light L1; an objective lens 4 with which a sample A is irradiated with the exciting light L1; a dichroic mirror 11 for branching the fluorescent light L2 emitted from the sample A from the exciting light L1; a 1st imaging lens 25 for imaging the branched fluorescent light L2; a 1st imaging device 7 arranged in the imaging position of the imaging lens 25; a transmissive-illumination light source 6 arranged opposite to the objective lens 4 across the sample A; a phase film and an absorption film 21 arranged at the pupil position of the objective lens 4; a 1st optical modulation member 18 arranged between the light source 6 and the sample A, and whose transmission peak stands outside a fluorescent wavelength band; a 2nd optical modulation member 22 for branching light L3 passing through the sample A and the objective lens 4 after passing through the 1st optical modulation member 18; a 2nd imaging lens 26 for imaging the branched light L3; and a 2nd imaging device 8 arranged at the imaging position of the 2nd imaging lens 26. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an observation system.

Conventionally, in the fields of medicine, biology, and the like, an epifluorescence microscope, a pupil modulation microscope (phase contrast microscope), and the like are used to observe the state of cells and tissues.
There is also known a microscope apparatus that switches between epifluorescence observation and phase difference observation (see, for example, Patent Document 1).

This microscope apparatus includes an excitation light source for fluorescence observation and a transmitted illumination light source for phase difference observation. The microscope apparatus generates fluorescence by causing excitation light emitted from the excitation light source to enter the sample. A fluorescence observation optical system for detection, and a phase difference observation optical system for performing phase difference observation by making light emitted from a transmitted illumination light source incident on a specimen are provided.
During fluorescence observation, the excitation light source is activated to cause excitation light to enter the sample, and the fluorescence emitted from the sample is observed with an eyepiece or an imaging device, while during phase difference observation, the transmitted illumination light source is activated, The light transmitted through the lens is observed with an eyepiece lens or an imaging device. Thereby, either fluorescence observation or phase difference observation can be selected and observed.
JP-A-8-15612 (FIG. 1 etc.)

However, the microscope apparatus of Patent Document 1 cannot perform fluorescence observation and phase difference observation at the same time.
Fluorescence observation is a method of observing a fluorescent signal of a fluorescent dye or fluorescent protein itself labeled on a sample, and is suitable for specifically detecting and visualizing an observation target. On the other hand, phase difference observation is an observation method that changes the phase distribution of a transparent object such as a cell into contrast, and is an observation method that can visualize the fine structure and external form in a cell. Thereby, the phase difference observation is suitable for observing a cell state such as cell activity. As a method of applying these observation methods, for example, when the state of a cell is confirmed when a change occurs in fluorescence emitted from the cell, phase difference observation may be performed in parallel with the fluorescence observation. In addition, when the fluorescence is emitted locally from a part of the specimen, the phase difference observation can be performed in parallel with the fluorescence observation even when observing from which position of the whole specimen the fluorescence is emitted. is there.

For these applications, in the microscope apparatus of Patent Document 1, since it is necessary to switch between the fluorescence observation optical system and the phase difference observation optical system, both the fluorescence observation and the phase difference observation are performed for a large amount of specimens in a short time. It was difficult to do with.
The present invention has been made in view of the above-described circumstances, and an object thereof is to provide an observation system capable of simultaneously performing pupil modulation observation such as phase difference observation and fluorescence observation.

In order to achieve the above object, the present invention provides the following means.
The present invention provides an excitation light source that emits excitation light, an objective lens that irradiates a specimen with excitation light emitted from the excitation light source, and a fluorescence that is emitted from the specimen and returns via the objective lens is branched from the excitation light. A dichroic mirror; a first imaging lens that forms an image of the branched fluorescence; a first imaging element that is disposed at an imaging position of the first imaging lens; and the objective lens with the sample interposed therebetween A transmission illumination light source disposed on the opposite side of the objective lens, a phase film and an absorption film disposed at the pupil position of the objective lens or a conjugate position thereof, and the transmission illumination light source disposed between the transmission illumination light source and the specimen. A first optical modulation member having a transmission peak outside the wavelength band, and a second optical modulation member that selectively passes through the first optical modulation member, enters the sample, and passes through the objective lens. And the second optical modulation unit Providing a second imaging lens for forming the light branched, an observation system and a second imaging device located on the imaging position of the second imaging lens by.

  According to the present invention, when the excitation light emitted from the excitation light source is irradiated to the specimen through the objective lens, the fluorescent substance contained in the specimen is excited to generate fluorescence. Fluorescence emitted from the specimen returns through the objective lens and is branched from the excitation light by the dichroic mirror. Then, the fluorescence branched from the excitation light is imaged by the first imaging lens, and is acquired as a fluorescence image by the first imaging device arranged at the imaging position.

  On the other hand, the light emitted from the transmitted illumination light source is allowed to pass through the first optical modulation member, so that only light having a wavelength different from the fluorescence wavelength is incident on the sample. The light transmitted through the sample and collected by the objective lens is passed through the phase film and the absorption film disposed at the pupil position of the objective lens or its conjugate position, so that the direct light is phase-modulated and Intensity modulated. After that, the light is incident on the second optical modulation member to be branched from other light having different wavelengths. Then, the branched light is imaged by the second imaging lens, and is acquired as a phase difference image by causing the diffracted light and the direct light to interfere with each other in the second imaging element disposed at the imaging position. It will be.

  As described above, according to the present invention, it is possible to provide separate imaging optical paths by differentiating the wavelength bands of the fluorescence observation and the phase difference observation, and to acquire the fluorescence image and the phase difference image at the same time. It becomes possible.

In the above invention, the dichroic mirror branches the excitation light and the light transmitted through the first optical modulation member from the fluorescence, and the second optical modulation member is disposed between the dichroic mirror and the excitation light source. It is good also as being done.
With this configuration, the excitation light from the excitation light source and the light from the transmitted illumination light source transmitted through the first optical modulation member are branched from the fluorescence by the dichroic mirror.

That is, the excitation light emitted from the excitation light source passes through the dichroic mirror or is reflected and enters the optical path toward the objective lens, and is irradiated onto the sample via the objective lens. The fluorescence emitted from the sample returns to the incident light path of the excitation light through the objective lens. Further, the light from the transmitted illumination light source passes through the sample after passing through the first optical modulation member, and enters the objective lens, and this also follows the incident light path of the excitation light. Then, by being incident on the dichroic mirror, the light that has passed through the first optical modulation member is directed toward the excitation light source and separated from the fluorescence. Thereafter, the light that has passed through the first optical modulation member is incident on the second optical modulation member disposed between the dichroic mirror and the excitation light source, and is branched from the incident optical path of the excitation light. Then, after passing through the second imaging lens, an image is taken by the second image sensor.
As described above, according to the present invention, the optical path for phase difference observation and the incident optical path of the excitation light can be partially used, and the overall length of the optical system can be shortened and the size can be reduced. .

Moreover, in the said invention, it is preferable that the said absorption film is arrange | positioned between the said 2nd optical modulation member and the said 2nd imaging lens.
Since the intensity of light decreases when passing through the absorption film, the light for phase difference observation is branched from the light for fluorescence observation by the second optical modulation member and then passed through the absorption film, thereby allowing fluorescence. A bright fluorescent image can be obtained by preventing a decrease in the intensity of light for observation.

In the above invention, the first optical modulation member may transmit light having a wavelength longer than the fluorescence wavelength.
When observing a living organism such as a cell as a specimen, there is an inconvenience that the specimen deteriorates early due to generation of active oxygen due to light irradiation. By making the phase difference observation light longer than the fluorescence wavelength, the specimen can be kept in a healthy state by irradiating the specimen with less toxic light.
In particular, the first optical modulation member may transmit light of 700 nm or more.

  In the above invention, the first optical modulation member transmits light in the first wavelength band longer than the fluorescence wavelength, and the first wavelength band is interposed between the objective lens and the dichroic mirror. A third optical modulation member that selectively branches the light having the above wavelength; and the second optical modulation member is disposed between the third optical modulation member and the second imaging lens. A focus detection device that detects a focus position using light having a wavelength longer than the first wavelength band branched by the second optical modulation member may be provided.

By the operation of the focus detection device, light having a wavelength longer than the first wavelength band is incident on the optical path toward the objective lens via the second optical modulation member and the third optical modulation member. Then, the focus detection light is applied to the specimen through the objective lens, and is reflected by the specimen to return to the same optical path and be detected by the focus detection device. Thereby, the focus position is detected. On the other hand, the light for phase difference observation is branched from the fluorescence by the third optical modulation member after passing through the sample, follows the same path as the optical path for focus detection, and the light for focus detection by the second optical modulation member. And the image is formed by the second imaging lens and imaged by the second imaging element.
As described above, according to the present invention, the optical path for phase difference observation and the optical path for focus detection can be partially used, and the overall length of the optical system can be shortened and the size can be reduced. .
Further, as the light for focus detection, the sample can be irradiated with light having less toxicity, and the sample can be maintained in a healthy state.

In the above invention, the absorption film may be disposed between the second optical modulation member and the second imaging lens.
A bright fluorescent image can be obtained by preventing a decrease in the intensity of light for fluorescence observation by the absorption film.
In the above invention, the first optical modulation member may transmit light of 700 nm or more.

  According to the present invention, fluorescence observation and pupil modulation observation such as phase difference observation can be performed at the same time, and both fluorescence observation and phase difference observation can be performed in a short time on a large number of specimens. Play.

Hereinafter, the observation system 1 according to the first embodiment of the present invention will be described with reference to FIG.
As shown in FIG. 1, the observation system 1 according to the present embodiment includes a stage 3 on which a transparent dish 2 in which a specimen A such as a cell is accommodated in a culture solution W, and a lower part of the stage 3. The objective lens 4 arranged in this manner, the excitation light source 5 for generating the excitation light L1 for irradiating the specimen A from below through the objective lens 4, and the transmission for irradiating the specimen A with light from above the specimen A An illumination light source 6, a first image sensor 7 that images fluorescence L <b> 2 generated in the specimen A, and a second image sensor 8 that images light L <b> 3 emitted from the transmitted illumination light source 6 and passed through the specimen A are provided. Yes.

In the incident optical path of the excitation light L 1 from the excitation light source 5 toward the objective lens 4, there is a light projecting optical system 9 that converts light from the excitation light source 5 into substantially parallel light, and light emitted from the excitation light source 5. An excitation filter 10 that generates the excitation light L1 and a dichroic mirror 11 that reflects and deflects the excitation light L1 generated by the excitation filter 10 and enters the optical path toward the objective lens 4 are arranged.
The light projecting optical system 9 includes, for example, a collector lens 12 that condenses light from the excitation light source 5, an aperture stop 13, a field stop 14, and condenser lenses 15 and 16. Is made into substantially parallel light having a predetermined light beam diameter and is incident on the dichroic mirror 11.

  Further, in the incident optical path from the transmitted illumination light source 6 toward the specimen A, only the collector lens 17 for condensing the light emitted from the transmitted illumination light source 6 and the light L3 in the wavelength band longer than the wavelength band of the fluorescence L2. And a ring diaphragm 19 having a ring-shaped slit that allows a part of the light L3 that has passed through the first optical modulation member 18 to pass therethrough. And a condenser lens 20 that forms an image of the light L3 focused by the ring diaphragm 19 on the specimen A.

  A phase film and an absorption film 21 are provided at the pupil position of the objective lens 4 to modulate the phase and intensity of the direct light transmitted without being diffracted in the specimen A. The phase film and the absorption film 21 are configured to pass the light diffracted in the sample A as it is without being modulated.

  Between the objective lens 4 and the dichroic mirror 11, light L3 having passed through the first optical modulation member 18 and having a wavelength band longer than the wavelength band of the fluorescence L2 is reflected and deflected. A second optical modulation member 22 that transmits light in the wavelength band of the fluorescence L2 and a barrier filter 23 that blocks light in the long wavelength band that has passed through the second optical modulation member 22 are disposed. Yes.

Between the dichroic mirror 11 and the first image sensor 7, the absorption filter 24 that blocks the excitation light L1 that has passed through the dichroic mirror 11 and the fluorescence L2 that has passed through the dichroic mirror 11 are collected and collected. And a first imaging lens 25 that forms an image on one imaging device 7.
Further, the light L 3 reflected by the second optical modulation member 22 is condensed between the second optical modulation member 22 and the second image sensor 8 and imaged on the second image sensor 8. A second imaging lens 26 is provided.

The operation of the observation system 1 according to the present embodiment configured as described above will be described below.
In order to observe the specimen A using the observation system 1 according to the present embodiment, the specimen A is arranged on the stage 3 and the excitation light source 5 and the transmitted illumination light source 6 are operated.
The light emitted from the excitation light source 5 is passed through the light projecting optical system 9 to be substantially parallel light, and is passed through the excitation filter 10, thereby becoming the excitation light L1 in a predetermined wavelength band and the dichroic mirror 11. It is made to enter. Since the dichroic mirror 11 is configured to reflect the excitation light L1 in this wavelength band, the excitation light L1 is reflected by the dichroic mirror 11 and enters the incident optical path toward the objective lens 4.

Then, the excitation light L1 passes through the barrier filter 23 and the second optical modulation member 22, and then enters the objective lens 4, is condensed by the objective lens 4, and is applied to the specimen A on the stage 3. .
Since there is a fluorescent material previously contained or injected in the specimen A, the fluorescent material is excited by the irradiated excitation light L1, and fluorescence L2 is emitted.

  The fluorescence L2 generated in the specimen A returns along the incident optical path of the excitation light L1, passes through the objective lens 4, the second optical modulation member 22, and the barrier filter 23, and is incident on the dichroic mirror 11. Since the dichroic mirror 11 is configured to pass the fluorescent light L2 without reflecting, the fluorescent light L2 passes through the dichroic mirror 11 and is collected by the first imaging lens 25, and the first imaging element 7. The image is captured by. Thereby, a fluorescence image is acquired by the first image sensor 7. Since the absorption filter 24 is arranged at the subsequent stage of the dichroic mirror 11, it is possible to prevent the excitation light L1 that has passed through the dichroic mirror 11 from entering the first image sensor 7 as stray light. Can do.

  On the other hand, the light emitted from the transmitted illumination light source 6 is collected by the collector lens 17 and then passed through the first optical modulation member 18. Thereby, only the light L3 having a wavelength band different from the wavelength band of the fluorescence L2, in particular, a wavelength band longer than the wavelength band of the fluorescence L2 (for example, 700 nm or more) is allowed to pass through the first optical modulation member 18. . Then, after passing through the ring diaphragm 19, the light is condensed by the condenser lens 20 and imaged on the specimen A on the stage 3.

  Part of the light L3 irradiated to the specimen A passes through the specimen A without being diffracted (direct light), and the other part diffracts and passes through the specimen A (diffracted light). The light is incident on the objective lens 4 disposed below. Since the phase film and the absorption film 21 are provided at the pupil position of the objective lens 4, the phase and intensity of only the direct light are modulated and are incident on the second optical modulation member 22 in a condensed state.

  Since the second optical modulation member 22 is configured to reflect only the light L3 having a wavelength band of 700 nm or more that has passed through the first optical modulation member 18, direct light and diffracted light transmitted through the specimen A are reflected. Is reflected and deflected. Then, it is imaged by the second imaging lens 26 and imaged by the second imaging element 8. Thereby, in the 2nd image pick-up element 8, the phase difference image obtained by interference of direct light and diffracted light will be acquired. Since the barrier filter 23 is disposed after the second optical modulation member 22, direct light and diffracted light that are transmitted without being reflected by the second optical modulation member 22 are blocked by the barrier filter 23. The stray light is prevented from reaching the first image sensor 7.

  As described above, according to the observation system 1 according to the present embodiment, the fluorescence observation L2 and the phase difference observation light L3 are branched by performing the fluorescence observation and the phase difference observation in different wavelength bands. Thus, the fluorescence image and the phase difference image can be acquired separately at the same time. Thereby, both the fluorescence image and the phase difference image can be acquired in a short time with respect to a large amount of specimens. As a result, when the fluorescence emitted from the cell changes, it becomes possible to accurately capture information on the activity of the cell.

  In the observation system 1 according to the present embodiment, the light L3 having a wavelength band of 700 nm or more longer than the wavelength band for fluorescence observation is used as the phase difference observation light L3 by the first optical modulation member 18. used. In a cell or the like used as the specimen A, active oxygen may be generated by light irradiation. However, if the light L3 having a wavelength band of 700 nm or more is irradiated, the toxicity is small, and the specimen A Can be observed while maintaining a healthy state for a long time.

  In the present embodiment, the phase film and the absorption film 21 are arranged at the pupil position of the objective lens 4, but instead of this, as shown in FIG. 2, the second optical modulation member 22 and the second film are arranged. A pupil relay optical system 27 is arranged between the imaging lens 26, the pupil position of the objective lens 4 is relayed, and the phase film and the absorption film are arranged at positions arranged in a conjugate relationship with the pupil position of the objective lens 4. 21 may be arranged. Alternatively, only the phase film may be disposed at the pupil position of the objective lens 4 and only the absorption film 21 may be disposed at the conjugate position. By doing so, the direct light for phase difference observation can be allowed to pass through the absorption film 21 after being separated from the fluorescence L2 by the second optical modulation member 22. Therefore, it is not necessary to pass the fluorescence L2 through the absorption film 21, and a bright fluorescence image can be acquired by preventing a decrease in the intensity of the fluorescence L2.

Next, an observation system 30 according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the observation system 1 according to the first embodiment described above, and the description thereof is omitted.

The observation system 30 according to the present embodiment is different from the observation system 1 according to the first embodiment in the arrangement of the second optical modulation member 22 and the wavelength characteristics of the dichroic mirror 31.
In the present embodiment, the second optical modulation member 22 is disposed between the light projecting optical system 9 and the excitation filter 10. The dichroic mirror 31 is designed to reflect the wavelength band of the excitation light L1 and the light L3 having a wavelength band of 700 nm or more longer than the wavelength band of the fluorescence L2 and transmit only the fluorescence L2. Yes.

The operation of the observation system 30 according to this embodiment configured as described above will be described below.
The light emitted from the excitation light source 5 is made substantially parallel light by passing through the light projecting optical system 9 and is incident on the second optical modulation member 22. Since the second optical modulation member 22 reflects only the light L3 having a wavelength band of 700 nm or more and allows the light having the other wavelength band to pass therethrough, the light emitted from the light projecting optical system 9 is the second optical component. The light is passed through the modulation member 22 and is incident on the excitation filter 10. The excitation filter 10 generates excitation light L 1 having a predetermined wavelength band.

  Thereafter, the excitation light L1 is reflected by the dichroic mirror 31 and irradiated onto the specimen A through the objective lens 4, and the fluorescence L2 generated in the specimen A is passed through the dichroic mirror 31 through the objective lens 4 to be the first. The point that an image is formed by the imaging lens 25 and is imaged by the first image sensor 7 is the same as that of the observation system 1 according to the first embodiment.

  On the other hand, the light L3 including direct light and diffracted light having a wavelength band of 700 nm or more, which is emitted from the transmitted illumination light source 6 and passed through the specimen A, is reflected by the dichroic mirror 31 to be incident on the excitation light L1. The light is separated from the excitation light L1 by being reflected by the second optical modulation member 22 at a midway position along the optical path to the light projecting optical system 9. Then, the phase film and the absorption film 21 arranged at the conjugate position of the pupil position of the objective lens 4 are passed through the pupil relay optical system 27 and imaged by the second imaging lens 26 to be second. The image pickup device 8 takes an image.

  According to the observation system 30 according to the present embodiment, similarly to the observation system 1 according to the first embodiment, a fluorescence image and a phase difference image can be acquired simultaneously. Furthermore, the observation system 30 according to the present embodiment partially reduces the total length of the optical path by minimizing the optical system by partially using the optical path of the phase difference observation light L3 as the incident optical path of the excitation light L1. Can be planned. Therefore, there is an advantage that the entire observation system 30 can be configured compactly.

Next, an observation system 40 according to a third embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to portions having the same configuration as the observation systems 1 and 30 according to the first and second embodiments described above, and the description thereof is omitted.

  In the observation system 40 according to the present embodiment, the first optical modulation member 18 is configured to transmit the light L1 in the first wavelength band of 700 nm to 780 nm. In addition, the observation system 40 according to the present embodiment reflects the light L1 and L4 in the wavelength band of 700 nm or more between the objective lens 4 and the dichroic mirror 11, and transmits the other light L2 and L3. The optical modulation member 41 is provided. Further, the observation system 40 according to the present embodiment detects the focus position by emitting the light L4 having a wavelength longer than the first wavelength band and detecting the light L4 reflected and returned from the sample A. A focus detection device 42 is provided.

In the present embodiment, the second optical modulation member 22 is disposed between the third optical modulation member 41 and the focus detection device 42. The second optical modulation member 22 is designed to reflect the light L1 in the first wavelength band of 700 nm to 780 nm and transmit the light L4 in the wavelength band of 780 nm or more.
Like the observation system 30 according to the second embodiment, the pupil relay optical system 27, the phase film and the absorption film 21, and the second optical modulation member 22 and the second imaging element 8 are arranged between the second optical modulation member 22 and the second imaging element 8. Two imaging lenses 26 are arranged.

The operation of the observation system 40 according to this embodiment configured as described above will be described below.
When the excitation light L1 directed to the objective lens 4 from the excitation light source 5 through the light projection optical system 9, the excitation filter 10, and the dichroic mirror 11 is irradiated onto the specimen A on the stage 3, it is generated in the specimen A. The fluorescence L2 passes through the objective lens 4, the third optical modulation member 41, and the dichroic mirror 11, is condensed by the first imaging lens 25, and is imaged by the first image sensor 7.

  On the other hand, the light emitted from the transmitted illumination light source 6 becomes only the light L3 in the first wavelength band of 700 nm to 780 nm by the first optical modulation member 18 and is applied to the sample A, and the direct light transmitted through the sample A. And the light L3 containing diffracted light is irradiated by the objective lens 4. The light L3 including the direct light and the diffracted light in the first wavelength band is reflected by the third optical modulation member 41 and separated from the fluorescence L2 on the way to the dichroic mirror 11, and the direction of the focus detection device 42 Oriented to. Further, the light L3 including the direct light and the diffracted light in the first wavelength band is reflected by the second optical modulation member 22 at a midpoint toward the focus detection device 42, and the pupil relay optical system 27, the phase film, and the absorption are reflected. The image is taken by the second image sensor 8 through the film 21 and the second imaging lens 26.

  Furthermore, according to the observation system 40 according to the present invention, the light L4 on the longer wavelength side than the first wavelength band emitted from the focus detection device 42, that is, the light L4 in the wavelength band of 780 nm or more, After passing through the optical modulation member 22, the light is reflected by the third optical modulation member 41, is incident on the objective lens 4, and is imaged on the specimen A by the objective lens 4. Then, the light L4 is reflected by the specimen A, returns along the same optical path, and is detected by the focus detection device 42. Accordingly, the focus detection device 42 can acquire a clear image by analyzing the detected light L4 to determine whether or not the focus position is appropriate and performing focus adjustment. .

According to the observation system 40 according to the present embodiment, the light L4 used for focus position detection is the light L4 on the longer wavelength side than the first wavelength band. It is possible to prevent the health of the specimen A from being hindered.
Further, by sharing a part of the optical path for phase difference observation with a part of the optical path for focus detection, there is an advantage that the optical path length can be shortened as a whole and the optical path can be made compact.

It is the schematic which shows the whole structure of the observation system which concerns on the 1st Embodiment of this invention. It is the schematic which shows the modification of the observation system of FIG. It is the schematic which shows the whole structure of the observation system which concerns on the 2nd Embodiment of this invention. It is the schematic which shows the whole structure of the observation system which concerns on the 3rd Embodiment of this invention.

Explanation of symbols

A specimen L1 excitation light L2 fluorescence L3, L4 light 1, 30, 40 observation system 4 objective lens 5 excitation light source 6 transmitted illumination light source 7 first imaging element 8 second imaging element 11, 31 dichroic mirror 18 first Optical modulation member 21 Absorbing film 22 Second optical modulation member 25 First imaging lens 26 Second imaging lens 41 Third optical modulation member 42 Focus detection device

Claims (8)

An excitation light source that emits excitation light;
An objective lens for irradiating the specimen with excitation light emitted from the excitation light source;
A dichroic mirror that diverges the fluorescence emitted from the specimen and returning through the objective lens from the excitation light;
A first imaging lens for imaging the branched fluorescence;
A first imaging element disposed at an imaging position of the first imaging lens;
A transmitted illumination light source disposed on the opposite side of the objective lens across the sample;
A phase film and an absorption film disposed at a pupil position of the objective lens or a conjugate position thereof; and
A first optical modulation member that is disposed between the transmitted illumination light source and the specimen and has a transmission peak outside the fluorescence wavelength range;
A second optical modulation member that selectively passes through the first optical modulation member and is incident on the sample and passes through the objective lens;
A second imaging lens that forms an image of the light branched by the second optical modulation member;
An observation system comprising: a second imaging device disposed at an imaging position of the second imaging lens. The dichroic mirror branches the excitation light and the light transmitted through the first optical modulation member from the fluorescence;
The observation system according to claim 1, wherein the second optical modulation member is disposed between the dichroic mirror and an excitation light source.   The observation system according to claim 1, wherein the absorption film is disposed between the second optical modulation member and the second imaging lens.   The observation system according to claim 3, wherein the first optical modulation member transmits light having a longer wavelength than the fluorescence wavelength.   The observation system according to claim 4, wherein the first optical modulation member transmits light of 700 nm or more. The first optical modulation member transmits light in a first wavelength band longer than the fluorescence wavelength;
Between the objective lens and the dichroic mirror, a third optical modulation member that selectively branches light having a wavelength of the first wavelength band or more,
The second optical modulation member is disposed between the third optical modulation member and the second imaging lens;
The observation system according to claim 1, further comprising a focus detection device that detects a focus position using light having a wavelength longer than the first wavelength band branched by the second optical modulation member.   The observation system according to claim 6, wherein the absorption film is disposed between the second optical modulation member and the second imaging lens.   The observation system according to claim 7, wherein the first optical modulation member transmits light of 700 nm or more.

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