JP2006174764A - Transmitted illumination device, microscope equipped wight the same and transmitted illumination method - Google Patents
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- 238000005286 illumination Methods 0.000 title claims abstract description 149
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- 238000009434 installation Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
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- C12M31/08—Means for providing, directing, scattering or concentrating light by conducting or reflecting elements located inside the reactor or in its structure
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Abstract
Description
本発明は、例えば、インキュベータなどの細胞培養環境下で、培養細胞の観察を行うために用いるそれを備えた顕微鏡、及び透過照明方法に関する。 The present invention relates to a microscope including the microscope used for observing cultured cells in a cell culture environment such as an incubator, and a transmitted illumination method.
近年、幹細胞培養など、細胞を大量に培養することが求められてきている。そして、従来、細胞を培養するための細胞培養装置として、インキュベータが使用されている。
インキュベータは、例えば、温度が約３７℃、湿度が約９５％、ＣＯ2の濃度が約５％等の一定の雰囲気に保たれた培養環境下で、無菌状態の環境を維持し、細胞を培養するように構成された装置である。
そして、従来、培養した細胞の標本を可視化して、細胞の活性や形態の観察を行うためには、インキュベータ内で培養した細胞標本をインキュベータから外部に取り出して、顕微鏡下で位相差観察等などの観察手法が用いられている。
しかしながら、このような観察手法では、培養細胞をインキュベータから外部に取り出すときに人手を介するため、コンタミの影響を受けることがあった。
In recent years, there has been a demand for culturing cells in large quantities, such as stem cell culture. And conventionally, an incubator has been used as a cell culture apparatus for culturing cells.
An incubator maintains a sterile environment and cultures cells in a culture environment maintained at a constant atmosphere such as a temperature of about 37 ° C., a humidity of about 95%, and a CO 2 concentration of about 5%. An apparatus configured to do this.
Conventionally, in order to visualize a cultured cell specimen and observe the activity and morphology of the cell, the cell specimen cultured in the incubator is taken out of the incubator and observed under a microscope, etc. The observation method is used.
However, in such an observation method, since the cultured cells are taken out manually from the incubator, they may be affected by contamination.
そこで、このような問題を解決するために、従来、培養細胞を外部に取り出すことなくインキュベータ内で観察するための技術が例えば、次の特許文献１において提案されている。
培養細胞を外部に取り出すことなくインキュベータ内で観察する場合、透過照明系の光源をインキュベータの内部に配置すると、光源が熱源として作用してしまうことになるため、培養環境の温度、湿度を一定に保つのが困難になる。
しかるに、特許文献１に記載の技術では、インキュベータ装置を改造して、光源を培養環境下には配置しない構成を採用している。
When observing the cultured cells in the incubator without taking them out, placing the light source of the transmission illumination system inside the incubator will cause the light source to act as a heat source, so the temperature and humidity of the culture environment should be kept constant. It becomes difficult to keep.
However, the technique described in Patent Document 1 employs a configuration in which the incubator apparatus is modified so that the light source is not placed in the culture environment.
しかしながら、特許文献１に記載の技術では、培養細胞の上方に配置された光源から照明光を照射し、培養細胞を透過した光を観察することができるように、透過照明光学系が配置されている。これでは、透過照明光学系の設置が煩雑となるので好ましくない。また、特許文献１に記載の技術では、光源を培養環境下には配置してはいないものの、インキュベータの筐体内に備えた構成となっている。このため、特許文献１に記載の技術においても、光源が熱源として機能し、培養環境の温度変化を招く要因となりやすい。 However, in the technique described in Patent Document 1, a transmission illumination optical system is disposed so that illumination light can be irradiated from a light source disposed above the cultured cells and light transmitted through the cultured cells can be observed. Yes. This is not preferable because the installation of the transmission illumination optical system becomes complicated. Moreover, although the light source is not arrange | positioned in culture | cultivation environment in the technique of patent document 1, it has the structure provided in the housing | casing of the incubator. For this reason, also in the technique described in Patent Document 1, the light source functions as a heat source, which tends to cause a temperature change in the culture environment.
本発明は、上記問題点に鑑みてなされたものであり、簡単な構成で、培養環境を一定に保ちながら、透過照明観察を行うことのできる透過照明装置、それを備えた顕微鏡、及び透過照明方法を提供することを目的とする。 The present invention has been made in view of the above problems, and has a simple configuration, a transmission illumination apparatus capable of performing transmission illumination observation while maintaining a constant culture environment, a microscope including the same, and transmission illumination It aims to provide a method.
上記目的を達成するため、本発明による透過照明装置は、筐体の外部に配置された照明用光源と、筐体の内部に配置された、前記照明用光源からの光を試料側に導く落射照明光学系と、前記落射照明光学系からの光を試料上に集光する対物レンズと、前記試料を挟んで前記対物レンズと対向する位置に配置されたコンデンサレンズと、前記コンデンサレンズの前側焦点位置近傍に配置された反射部材とを備え、前記反射部材からの反射光を用いて前記試料を透過照明するようにしたことを特徴としている。 In order to achieve the above object, a transmission illumination device according to the present invention includes an illumination light source disposed outside a housing, and an epi-illumination disposed inside the housing for guiding light from the illumination light source to the sample side. An illumination optical system; an objective lens that condenses the light from the epi-illumination optical system on the sample; a condenser lens disposed at a position facing the objective lens across the specimen; and a front focal point of the condenser lens And a reflecting member arranged in the vicinity of the position, and the sample is transmitted and illuminated using reflected light from the reflecting member.
また、本発明の透過照明装置においては、前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置のいずれかに、リングスリットを配置するとともに、前記対物レンズの瞳位置に、位相板を配置するのが好ましい。 In the transmission illumination device of the present invention, a ring slit is disposed at any one of a position conjugate with the pupil position of the objective lens and a front focal position of the condenser lens in the incident light optical system, and A phase plate is preferably disposed at the pupil position of the objective lens.
また、本発明の透過照明装置においては、前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置の両方に、リングスリットを配置するとともに、前記対物レンズの瞳位置に、位相板を配置するのが好ましい。 In the transmission illumination device of the present invention, a ring slit is disposed both at a position conjugate with the pupil position of the objective lens and at a front focal position of the condenser lens in the incident light optical system, and the objective It is preferable to arrange a phase plate at the pupil position of the lens.
また、本発明の透過照明装置においては、前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置のいずれかに、リングスリットを配置するとともに、前記対物レンズの瞳位置と共役となる位置に、位相板を配置するのが好ましい。 In the transmission illumination device of the present invention, a ring slit is disposed at any one of a position conjugate with the pupil position of the objective lens and a front focal position of the condenser lens in the incident light optical system, and It is preferable to arrange the phase plate at a position conjugate with the pupil position of the objective lens.
また、本発明の透過照明装置においては、前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置の両方に、リングスリットを配置するとともに、前記対物レンズの瞳位置と共役となる位置に、位相板を配置するのが好ましい。 In the transmission illumination device of the present invention, a ring slit is disposed both at a position conjugate with the pupil position of the objective lens and at a front focal position of the condenser lens in the incident light optical system, and the objective It is preferable to arrange the phase plate at a position conjugate with the pupil position of the lens.
また、本発明の透過照明装置においては、前記試料と前記反射部材との間に、λ／４板を配置し、落射照明光の光路上に、偏光子を配置し、透過照明光の光路上に、検光子を配置するのが好ましい。 In the transmission illumination device of the present invention, a λ / 4 plate is disposed between the sample and the reflecting member, a polarizer is disposed on the optical path of the epi-illumination light, and the optical path of the transmission illumination light is In addition, it is preferable to arrange an analyzer.
また、本発明の透過照明装置においては、前記筐体の内部に複数の試料を培養するための細胞培養庫を有し、前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、前記照明位置に移動した試料から前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されているのが好ましい。 In the transmission illumination device of the present invention, the housing has a cell culture chamber for culturing a plurality of samples inside the casing, and the cell culture chamber illuminates a predetermined sample among the plurality of samples. It is preferable that an optical member that is provided on the optical path from the sample that has been moved to the illumination position to the reflection member is disposed inside the cell culture chamber.
また、本発明の透過照明装置においては、前記筐体の内部に複数の試料を培養するための細胞培養庫を有し、前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、前記位相板から前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されているのが好ましい。 In the transmission illumination device of the present invention, the housing has a cell culture chamber for culturing a plurality of samples inside the casing, and the cell culture chamber illuminates a predetermined sample among the plurality of samples. It is preferable that an optical member that is provided on the optical path from the phase plate to the reflecting member is provided inside the cell culture chamber.
また、本発明の透過照明装置においては、前記筐体の内部に複数の試料を培養するための細胞培養庫を有し、前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、前記対物レンズから前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されているのが好ましい。 In the transmission illumination device of the present invention, the housing has a cell culture chamber for culturing a plurality of samples inside the casing, and the cell culture chamber illuminates a predetermined sample among the plurality of samples. It is preferable that an optical member that is provided on the optical path from the objective lens to the reflection member is provided inside the cell culture chamber, and includes a moving means that can move to a position.
また、本発明による顕微鏡は、上記本発明によるいずれかの透過照明装置を備えている。 A microscope according to the present invention includes any one of the above-described transmission illumination devices according to the present invention.
また、本発明による顕微鏡は、上記本発明によるいずれかの透過照明装置と、前記筐体の内部に、前記透過照明装置を介して透過照明された前記試料の像を観察する観察光学系を備えている。 A microscope according to the present invention includes any of the above-described transmission illumination devices according to the present invention, and an observation optical system that observes an image of the sample that is transmitted and illuminated through the transmission illumination device inside the casing. ing.
また、本発明による顕微鏡は、筐体の外部に配置された照明用光源と、筐体の内部に配置された、前記照明用光源からの光を試料側に導く落射照明光学系と、前記落射照明光学系からの光を試料上に集光する対物レンズと、前記試料を挟んで前記対物レンズと対向する位置に配置されたコンデンサレンズと、前記コンデンサレンズの前側焦点位置近傍に配置された反射部材とを備え、前記反射部材からの反射光を用いて前記試料を透過照明するようにした透過照明装置と、前記筐体の内部に、前記透過観察装置を介して透過照明された前記試料の像を観察する観察光学系を備え、前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置のいずれかに、リングスリットを配置するとともに、前記観察光学系内において前記対物レンズの瞳位置と共役となる位置に、位相板を配置したことを特徴としている。 The microscope according to the present invention includes an illumination light source disposed outside the housing, an epi-illumination optical system disposed inside the housing for guiding light from the illumination light source to the sample side, and the epi-illumination. An objective lens for condensing the light from the illumination optical system on the sample, a condenser lens arranged at a position facing the objective lens across the specimen, and a reflection arranged near the front focal position of the condenser lens A transmission illuminating device configured to transmit and illuminate the sample using reflected light from the reflecting member, and the sample transmitted through the transmission observation device to the inside of the housing. An observation optical system for observing an image, and a ring slit is disposed at one of a position conjugate with the pupil position of the objective lens and a front focal position of the condenser lens in the incident light optical system. , The pupil position conjugate with a position of said objective lens in said observation optical system is characterized in that a phase plate.
また、本発明による顕微鏡は、筐体の外部に配置された照明用光源と、筐体の内部に配置された、前記照明用光源からの光を試料側に導く落射照明光学系と、前記落射照明光学系からの光を試料上に集光する対物レンズと、前記試料を挟んで前記対物レンズと対向する位置に配置されたコンデンサレンズと、前記コンデンサレンズの前側焦点位置近傍に配置された反射部材とを備え、前記反射部材からの反射光を用いて前記試料を透過照明するようにした透過照明装置と、前記筐体の内部に、前記透過観察装置を介して透過照明された前記試料の像を観察する観察光学系を備え、前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置の両方に、リングスリットを配置するとともに、前記観察光学系内において前記対物レンズの瞳位置と共役となる位置に、位相板を配置したことを特徴としている。 The microscope according to the present invention includes an illumination light source disposed outside the housing, an epi-illumination optical system disposed inside the housing for guiding light from the illumination light source to the sample side, and the epi-illumination. An objective lens for condensing the light from the illumination optical system on the sample, a condenser lens arranged at a position facing the objective lens across the specimen, and a reflection arranged near the front focal position of the condenser lens A transmission illuminating device configured to transmit and illuminate the sample using reflected light from the reflecting member, and the sample transmitted through the transmission observation device to the inside of the housing. An observation optical system for observing an image is provided, and ring slits are disposed at both the position that is conjugate with the pupil position of the objective lens in the incident light optical system, and the front focal position of the condenser lens, and In the serial observation optical system at the pupil position conjugate with a position of the objective lens is characterized in that a phase plate.
また、本発明の顕微鏡においては、前記試料と前記反射部材との間に、λ／４板を配置し、落射照明光の光路上に、偏光子を配置し、透過照明光の光路上に、検光子を配置するのが好ましい。 In the microscope of the present invention, a λ / 4 plate is disposed between the sample and the reflecting member, a polarizer is disposed on the optical path of the epi-illumination light, and on the optical path of the transmitted illumination light. An analyzer is preferably arranged.
また、本発明の顕微鏡においては、前記筐体の内部に複数の試料を培養するための細胞培養庫を有し、前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、前記照明位置に移動した試料から前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されているのが好ましい。 Moreover, in the microscope of the present invention, the housing has a cell culture chamber for culturing a plurality of samples, and the cell culture chamber has a predetermined sample among the plurality of samples at an illumination position. It is preferable that an optical member provided on the optical path from the sample that has been moved to the illumination position to the reflecting member is disposed inside the cell culture chamber.
また、本発明の顕微鏡においては、前記筐体の内部に複数の試料を培養するための細胞培養庫を有し、前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、前記対物レンズから前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されているのが好ましい。 Moreover, in the microscope of the present invention, the housing has a cell culture chamber for culturing a plurality of samples, and the cell culture chamber has a predetermined sample among the plurality of samples at an illumination position. It is preferable that an optical member provided with movable means and arranged on the optical path from the objective lens to the reflecting member is arranged inside the cell culture chamber.
また、本発明による透過照明方法は、筐体の外部に配置した照明用光源からの光を用いて、筐体の内部に配置した試料を落射照明し、落射照明したときに前記試料を透過した落射光を反射部材で反射し、反射した光で前記試料を透過照明することを特徴としている。 Further, the transmission illumination method according to the present invention uses the light from the illumination light source arranged outside the casing to illuminate the sample arranged inside the casing and transmits the sample when the specimen is illuminated. The reflected light is reflected by a reflecting member, and the sample is transmitted and illuminated with the reflected light.
また、本発明による透過照明方法は、上記いずれかの本発明の透過照明装置を介して、前記細胞培養庫内の試料を落射照明し、落射照明したときに前記試料を透過した落射光を前記反射部材で反射し、反射した光で前記試料を透過照明することを特徴としている。 In addition, the transmitted illumination method according to the present invention is configured such that the sample in the cell culture box is incidentally illuminated via the transmitted illumination apparatus according to any one of the present invention, and the incident light transmitted through the sample when the incident illumination is applied is described above. The sample is reflected by a reflecting member, and the sample is transmitted and illuminated with the reflected light.
本発明によれば、簡単な構成で、培養環境を一定に保ちながら、透過照明観察を行うことのできる透過照明装置、それを備えた顕微鏡、及び透過照明方法が得られる。 According to the present invention, it is possible to obtain a transmission illumination apparatus, a microscope including the transmission illumination apparatus, and a transmission illumination method capable of performing transmission illumination observation with a simple configuration while keeping the culture environment constant.
図１は本発明の第一実施形態にかかる透過照明装置を備えた顕微鏡の概略構成図である。
第一実施形態の顕微鏡は、筐体１の内部に、細胞培養庫２と、透過照明光学系３と、結像光学系４を有している。また、筐体１の外部に、照明用光源５と、撮像素子６と、画像処理装置７を備えている。
細胞培養庫２は、温度が３７℃、湿度が９５％の雰囲気でもって培養環境が一定に保たつように培養環境調整手段（図示省略）を備えた筐体２ａからなる。筐体２ａの内部には、培養標本（試料）１０を培養するシャーレ１１1，１１2，・・・１１nを複数個（ｎ個）備えている。また、筐体２ａは、所望のシャーレを照明光の照射位置に移動可能な移動手段を有する移動ステージ１２を備えている。なお、培養環境調整手段、移動手段は、既知の公知技術を用いて構成することができる。
FIG. 1 is a schematic configuration diagram of a microscope including a transmission illumination device according to the first embodiment of the present invention.
The microscope according to the first embodiment includes a cell culture chamber 2, a transmission illumination optical system 3, and an imaging optical system 4 inside a housing 1. In addition, an illumination light source 5, an image sensor 6, and an image processing device 7 are provided outside the housing 1.
The cell culture chamber 2 is composed of a housing 2a provided with culture environment adjusting means (not shown) so that the culture environment is kept constant in an atmosphere having a temperature of 37 ° C. and a humidity of 95%. Inside the housing 2a, a plurality (n pieces) of petri dishes 11 1 , 11 2 ,... 11 n for culturing the culture specimen (sample) 10 are provided. The housing 2a includes a moving stage 12 having moving means capable of moving a desired petri dish to the irradiation position of the illumination light. The culture environment adjusting means and the moving means can be configured using a known publicly known technique.
透過照明光学系３は、落射照明光学系３ａと、対物レンズ３ｂと、コンデンサレンズ３ｃと、反射部材３ｄを有している。
落射照明光学系３ａは、明るさ絞り３ａ1と、レンズ３ａ2と、ハーフミラー３ａ3を有し、照明用光源５からの光を対物レンズ３ｂに導くように構成されている。
対物レンズ３ｂは、落射照明光学系３ａからの光を細胞培養庫２の筐体２ａの内部において照射位置に位置する培養標本（試料）１０上に集光する機能を備えている。
コンデンサレンズ３ｃは、細胞培養庫２の筐体２ａの内部において、培養標本１０を挟んで対物レンズ３ｂと対向する位置に配置されている。
反射部材３ｄは、細胞培養庫２の筐体２ａの内部において、コンデンサレンズ３ｃの前側焦点位置近傍に配置されている。
そして、反射部材３ｄは、落射照明光学系３ａ、対物レンズ３ｂを経た照明用光源５からの光を反射して、培養標本１０を透過照明するようになっている。
結像光学系４は、ハーフミラー３ａ3と撮像素子６との間に配置された、偏向ミラー４ａと結像レンズ４ｂを有して構成されている。
画像処理装置７は、パソコンなどの演算処置装置で構成されている。
The transmission illumination optical system 3 includes an epi-illumination optical system 3a, an objective lens 3b, a condenser lens 3c, and a reflection member 3d.
Epi-illumination optical system 3a includes a 3a 1 aperture stop, a lens 3a 2, has a half mirror 3a 3, and the light from the illumination light source 5 is configured to direct the objective lens 3b.
The objective lens 3b has a function of condensing the light from the epi-illumination optical system 3a on the culture specimen (sample) 10 located at the irradiation position inside the housing 2a of the cell culture chamber 2.
The condenser lens 3c is disposed inside the housing 2a of the cell culture chamber 2 at a position facing the objective lens 3b with the culture specimen 10 interposed therebetween.
The reflecting member 3d is disposed in the vicinity of the front focal position of the condenser lens 3c inside the housing 2a of the cell culture chamber 2.
The reflecting member 3d reflects the light from the illumination light source 5 that has passed through the epi-illumination optical system 3a and the objective lens 3b to transmit and illuminate the culture specimen 10.
The imaging optical system 4 includes a deflection mirror 4a and an imaging lens 4b disposed between the half mirror 3a 3 and the image sensor 6.
The image processing device 7 is configured by an arithmetic processing device such as a personal computer.
さらに、第一実施形態の透過照明装置を備えた顕微鏡では、リングスリット８が、コンデンサレンズ３ｃの前側焦点位置に配置されるとともに、位相板９が、対物レンズ３ｂの瞳位置に配置されている。 Furthermore, in the microscope provided with the transmission illumination device of the first embodiment, the ring slit 8 is disposed at the front focal position of the condenser lens 3c, and the phase plate 9 is disposed at the pupil position of the objective lens 3b. .
このように構成された第一実施形態の透過照明装置を備えた顕微鏡では、筐体１の外部に設けられた照明用光源５から発した光が、明るさ絞り３ａ1、レンズ３ａ2を経てハーフミラー３ａ3に入射する。そして、ハーフミラー３ａ3を透過した光が、位相板９、対物レンズ３ｂを経て、細胞培養庫２の筐体２ａの内部において照射位置に位置するシャーレの中の培養標本１０を照射する。ここで、培養標本１０を透過した光が、コンデンサレンズ３ｃを経て、リングスリット８に入射し、リングスリット８を通過した光が、反射部材３ｄに入射する。反射部材３ｄで反射した光は、逆の光路を辿って、培養標本１０を照射する。ここで、培養標本１０を透過した光が、対物レンズ３ｂ、位相板９を経て、ハーフミラー３ａ3に入射する。そして、ハーフミラー３ａ3を透過した光が、偏向ミラー４ａ、結像レンズ４ｂを経て撮像素子６の撮像面上で結像し、撮像素子６で撮像される。撮像素子６で撮像された培養標本１０の透過観察像の電気信号は画像処理装置７を介して所定の画像処理が施された後、画像処理装置７に一体又は別体のモニタ等に表示される。 In the microscope having the transmission illumination device of the first embodiment configured as described above, light emitted from the illumination light source 5 provided outside the housing 1 passes through the aperture stop 3a 1 and the lens 3a 2. The light enters the half mirror 3a 3 . The light transmitted through the half mirror 3a 3 is, the phase plate 9, through the objective lens 3b, irradiating the cultured specimen 10 in the petri dish located at the irradiation position in the interior of the housing 2a of the cell culture chamber 2. Here, the light that has passed through the culture specimen 10 enters the ring slit 8 through the condenser lens 3c, and the light that has passed through the ring slit 8 enters the reflecting member 3d. The light reflected by the reflecting member 3d follows the reverse optical path and irradiates the culture specimen 10. Here, the light transmitted through the culture specimen 10 enters the half mirror 3a 3 through the objective lens 3b and the phase plate 9. Then, the light transmitted through the half mirror 3a 3 forms an image on the imaging surface of the image sensor 6 through the deflection mirror 4a and the imaging lens 4b, and is imaged by the image sensor 6. The electrical signal of the transmission observation image of the culture specimen 10 imaged by the imaging element 6 is subjected to predetermined image processing via the image processing device 7 and then displayed on the image processing device 7 on an integral or separate monitor. The
第一実施形態の透過観察照明装置を備えた顕微鏡によれば、照明用光源５を顕微鏡の筐体１の外部に配置したので、照明用光源５が熱源として細胞培養庫２に影響を与えることがない。また、落射照明光の透過光を反射させることによって透過照明光として使用するようにしたので、落射照明と透過照明の２つの光源を配置する必要がなくて済み、照明系の構成を簡素化することができる。また、透過照明光学系を構成するために細胞培養庫２の筐体２ａの内部に配置すべき光学部材を極力少なくすることができ、構成を簡素化できる。 According to the microscope equipped with the transmission observation illumination device of the first embodiment, since the illumination light source 5 is arranged outside the microscope housing 1, the illumination light source 5 affects the cell culture chamber 2 as a heat source. There is no. Further, since the transmitted light of the epi-illumination light is reflected to be used as the trans-illumination light, it is not necessary to arrange two light sources of the epi-illumination and the trans-illumination, and the configuration of the illumination system is simplified. be able to. Further, the number of optical members that should be arranged inside the housing 2a of the cell culture cabinet 2 in order to configure the transmitted illumination optical system can be reduced as much as possible, and the configuration can be simplified.
なお、図１では、位相差観察を行う構成を示したが、第一実施形態の透過照明装置を備えた顕微鏡は、それ以外の顕微鏡観察にも適用可能である。
例えば、通常の明視野観察を行う場合には、リングスリット８と位相板９を取り外した構成とすればよい。
また、例えば、ホフマンモジュレーションコントラスト（ＨＭＣ）観察を行う場合には、リングスリット８をＨＭＣ観察用スリットに取り替えるとともに、位相板９をＨＭＣモジュレータに取り替えた構成とすればよい。
また、例えば、微分干渉観察を行う場合には、リングスリット８と位相板９を取り外し、ハーフミラー３ａ3のレンズ３ａ2側に偏光子とＤＩＣプリズムを配置すると共に、ハーフミラー３ａ3の偏向ミラー４ａ側に検光子とＤＩＣプリズムを配置すればよい。
また、第一実施形態の透過照明装置を備えた顕微鏡は、培養標本１ａに対して偏射照明となる構成であってもよい。
さらに、ハーフミラー３ａ3をユニット化して着脱可能とするとよい。その場合、他のユニットとしてハーフミラー３ａ3に所望の蛍光以外の波長をカットするバリアフィルタを備えたユニットや、さらには、これらに加えてハーフミラー３ａ3のそれぞれ異なる側に偏光子と検光子を配置したユニット等を構成し、それぞれのユニットの着脱に応じて、通常光の観察と蛍光観察、さらには偏光による観察とを切り替え可能に構成してもよい。
In addition, although the structure which performs a phase difference observation was shown in FIG. 1, the microscope provided with the transmission illumination apparatus of 1st embodiment is applicable also to other microscope observation.
For example, when performing normal bright field observation, the ring slit 8 and the phase plate 9 may be removed.
Further, for example, when performing Hoffman modulation contrast (HMC) observation, the ring slit 8 may be replaced with an HMC observation slit, and the phase plate 9 may be replaced with an HMC modulator.
For example, when performing a differential interference observation, remove the ring slit 8 and the phase plate 9, together with arranging the polarizer and the DIC prism lens 3a 2 side of the half mirror 3a 3, the deflecting mirror of the half mirror 3a 3 An analyzer and a DIC prism may be disposed on the 4a side.
Moreover, the microscope provided with the transmission illumination device of the first embodiment may be configured to be incident illumination on the culture specimen 1a.
Further, the half mirror 3a 3 is preferably unitized and detachable. In that case, a unit provided with a barrier filter that cuts the wavelength other than the desired fluorescence in the half mirror 3a 3 as another unit, and in addition to these, a polarizer and an analyzer on different sides of the half mirror 3a 3 May be configured so that normal light observation, fluorescence observation, and observation by polarized light can be switched in accordance with the attachment and detachment of each unit.
図２は本発明の第二実施形態にかかる透過照明装置を備えた顕微鏡の概略構成図である。
第二実施形態の顕微鏡は、リングスリット８’が、対物レンズ２ｂの瞳位置と共役となる位置である図１における明るさ絞り３ａ1の位置に配置されている。
第二実施形態の透過照明装置を備えた顕微鏡によれば、細胞培養庫２の筐体２ａの内部に設ける光学部材を、図１におけるリングスリット８の分だけ減らすことができ、細胞培養庫２の筐体２ａの環境をより安定させ易くなる。また、細胞培養庫２の筐体２ａの内部を照射する光をリングスリット８’を介して予め減らすことができるので、培養標本を照射する光を透過観察のために必要な最小限の光量に抑えることができ、フレアの発生を抑えやすくすることができるとともに、培養標本を照射することによる熱の影響を極力少なくすることができる。
その他の構成及び作用効果は第一実施形態の透過照明装置を備えた顕微鏡とほぼ同じである。
FIG. 2 is a schematic configuration diagram of a microscope provided with a transmission illumination device according to the second embodiment of the present invention.
In the microscope of the second embodiment, the ring slit 8 ′ is disposed at the position of the aperture stop 3a 1 in FIG. 1, which is a position conjugate with the pupil position of the objective lens 2b.
According to the microscope provided with the transmission illumination device of the second embodiment, the optical members provided in the housing 2a of the cell culture cabinet 2 can be reduced by the ring slit 8 in FIG. It becomes easier to stabilize the environment of the housing 2a. In addition, since the light that irradiates the inside of the housing 2a of the cell culture chamber 2 can be reduced in advance through the ring slit 8 ', the light that irradiates the culture specimen can be reduced to the minimum amount necessary for transmission observation. It is possible to suppress the occurrence of flare, and it is possible to reduce the influence of heat by irradiating the culture specimen as much as possible.
Other configurations and operational effects are almost the same as those of the microscope including the transmission illumination device of the first embodiment.
図３は本発明の第三実施形態にかかる透過照明装置を備えた顕微鏡の概略構成図である。
第三実施形態の顕微鏡は、リングスリット８が、コンデンサレンズ３ｃの前側焦点位置に配置されると共に、リングスリット８’が、対物レンズ２ｂの瞳位置を共役となる位置である図１における明るさ絞り３ａ1の位置に配置されている。
第三実施形態の透過照明装置を備えた顕微鏡によれば、リングスリット８’を介して、細胞培養庫２の筐体２ａの内部を照射する光を予め減らすことができるので、培養標本を照射する光を透過観察のために必要な最小限の光量に抑えることができ、培養標本を照射することによる熱の影響を極力少なくすることができる。しかも、リングスリットを２つ備えたので、フレアの発生をより一層抑えやすくすることができる。
その他の構成及び作用効果は第一実施形態の透過照明装置を備えた顕微鏡とほぼ同じである。
FIG. 3 is a schematic configuration diagram of a microscope including a transmission illumination device according to the third embodiment of the present invention.
In the microscope of the third embodiment, the ring slit 8 is arranged at the front focal position of the condenser lens 3c, and the ring slit 8 'is a position where the pupil position of the objective lens 2b is conjugate to the brightness in FIG. It is arranged at the position of the diaphragm 3a 1 .
According to the microscope equipped with the transmission illumination device of the third embodiment, the light that irradiates the inside of the housing 2a of the cell culture cabinet 2 can be reduced in advance through the ring slit 8 ′, so that the culture specimen is irradiated. Can be suppressed to the minimum amount of light necessary for transmission observation, and the influence of heat by irradiating the culture specimen can be minimized. In addition, since two ring slits are provided, the occurrence of flare can be further easily suppressed.
Other configurations and operational effects are almost the same as those of the microscope including the transmission illumination device of the first embodiment.
図４は本発明の第四実施形態にかかる透過照明装置を備えた顕微鏡の概略構成図である。
第四実施形態の顕微鏡は、図３の構成に加えて、培養標本１０と反射部材３ｄとの間にλ／４板１３を配置して構成されている(図４では一例として、λ／４板１３は、リングスリット８とコンデンサレンズ３ｃとの間に配置されている)。また、ハーフミラー３ａ3のレンズ３ａ2側及び偏向ミラー４ａ側に偏光子１４及び検光子１５をそれぞれ配置して構成されており、位相差偏光観察を行うことができるようになっている。
FIG. 4 is a schematic configuration diagram of a microscope provided with a transmission illumination device according to the fourth embodiment of the present invention.
The microscope of the fourth embodiment is configured by arranging a λ / 4 plate 13 between the culture specimen 10 and the reflecting member 3d in addition to the configuration of FIG. 3 (in FIG. 4, as an example, λ / 4 The plate 13 is disposed between the ring slit 8 and the condenser lens 3c). Further, the polarizer 14 and the analyzer 15 are respectively arranged on the lens 3a 2 side and the deflection mirror 4a side of the half mirror 3a 3 so that phase difference polarization observation can be performed.
このように構成された第四実施形態の透過照明装置を備えた顕微鏡では、筐体１の外部に設けられた照明用光源５から発した光が、スリット８’、レンズ３ａ2を経て、偏光子１３に入射し、一方の直線偏光成分の光が偏光子１４を通過してハーフミラー３ａ3に入射する。そして、ハーフミラー３ａ3を透過した光が位相板９、対物レンズ３ｂを経て、細胞培養庫２の筐体２ａの内部において照射位置に位置するシャーレの中の培養標本１０を照射する。ここで、培養標本１０を透過した光が、コンデンサレンズ３ｃを経て、λ／４板１３に入射し、λ／４板１３を介して円偏光成分に偏光されて、リングスリット８に入射する。そして、リングスリット８を通過した光が、反射部材３ｄに入射する。反射部材３ｄで反射した光は、逆の光路を辿って、培養標本１０を照射する。その際、円偏光成分が、λ／４板１３を介して、上記一方の直線偏光成分に対して垂直な他方の直線偏光成分の光に偏光される。ここで、培養標本１０を透過した光が、対物レンズ３ｂ、位相板９を経て、ハーフミラー３ａ3に入射する。そして、ハーフミラー３ａ3を透過した光が、検光子１５に入射する。このとき、他方の直線偏光成分の光に偏光された光は検光子１５を透過し、偏向ミラー４ａ、結像レンズ４ｂを経て撮像素子６の撮像面上で結像し、撮像素子６で撮像される。撮像素子６で撮像された培養標本１０の透過観察像の電気信号は画像処理装置７を介して所定の画像処理が施された後、画像処理装置７に一体又は別体のモニタ等に表示される。 In the microscope having the transmission illumination device of the fourth embodiment configured as described above, light emitted from the illumination light source 5 provided outside the housing 1 is polarized through the slit 8 ′ and the lens 3a 2. The light enters one of the polarizers 13, and one linearly polarized light component passes through the polarizer 14 and enters the half mirror 3 a 3 . Then, a half mirror 3a 3 light phase plate 9 having passed through the, through the objective lens 3b, irradiating the cultured specimen 10 in the petri dish located at the irradiation position in the interior of the housing 2a of the cell culture chamber 2. Here, the light transmitted through the culture specimen 10 enters the λ / 4 plate 13 through the condenser lens 3 c, is polarized into a circularly polarized component through the λ / 4 plate 13, and enters the ring slit 8. And the light which passed the ring slit 8 injects into the reflection member 3d. The light reflected by the reflecting member 3d follows the reverse optical path and irradiates the culture specimen 10. At that time, the circularly polarized light component is polarized by the other linearly polarized light component perpendicular to the one linearly polarized light component via the λ / 4 plate 13. Here, the light transmitted through the culture specimen 10 enters the half mirror 3a 3 through the objective lens 3b and the phase plate 9. Then, the light that has passed through the half mirror 3a 3 enters the analyzer 15. At this time, the light polarized into the light of the other linearly polarized light component passes through the analyzer 15, forms an image on the image pickup surface of the image pickup device 6 through the deflection mirror 4 a and the imaging lens 4 b, and picks up an image with the image pickup device 6 Is done. The electrical signal of the transmission observation image of the culture specimen 10 imaged by the imaging element 6 is subjected to predetermined image processing via the image processing device 7 and then displayed on the image processing device 7 on an integral or separate monitor. The
第四実施形態の透過照明装置を備えた顕微鏡によれば、偏光子１４及び検光子１５を介して所定の偏光成分のみを通過させるようにしたので、フレア光をより一層除去することができる。
その他の構成及び作用効果は第三実施形態の透過照明装置を備えた顕微鏡とほぼ同じである。
According to the microscope provided with the transmission illumination device of the fourth embodiment, only the predetermined polarization component is allowed to pass through the polarizer 14 and the analyzer 15, so that flare light can be further removed.
Other configurations and operational effects are substantially the same as those of the microscope including the transmission illumination device of the third embodiment.
図５は本発明の第五実施形態にかかる透過照明装置を備えた顕微鏡の概略構成図である。
第五実施形態の顕微鏡は、図４に示した第四実施形態の構成に加えて、筐体１の外部に結像レンズ４ｂで結像した中間像をリレーするためのリレー光学系１６を備えるとともに、位相板９を、対物レンズ３ｂの瞳位置ではなく、リレー光学系１６を構成するレンズ１６ａ，１６ｂの間に配置して構成している。
第五実施形態の顕微鏡によれば、位相板９を筐体１の外部に配置することになるので、培養標本１０を透過照明する途中での光量のロスがない。このため、より一層良好な透過観察像を観察することができる。
その他の構成は、第四実施形態の透過照明装置を備えた顕微鏡とほぼ同じである。
FIG. 5 is a schematic configuration diagram of a microscope provided with a transmission illumination device according to a fifth embodiment of the present invention.
In addition to the configuration of the fourth embodiment shown in FIG. 4, the microscope of the fifth embodiment includes a relay optical system 16 for relaying an intermediate image formed by the imaging lens 4 b outside the housing 1. At the same time, the phase plate 9 is arranged not between the pupil position of the objective lens 3b but between the lenses 16a and 16b constituting the relay optical system 16.
According to the microscope of the fifth embodiment, the phase plate 9 is arranged outside the housing 1, so there is no loss of light quantity during the transmission illumination of the culture specimen 10. For this reason, an even better transmission observation image can be observed.
Other configurations are almost the same as those of the microscope including the transmission illumination device of the fourth embodiment.
本発明の透過照明装置、それを備えた顕微鏡、及び透過照明方法は、医学、生物学、薬学、農学等、細胞等の培養物を観察することが分野において有用である。 The transmitted illumination apparatus of the present invention, the microscope equipped with the transmitted illumination apparatus, and the transmitted illumination method are useful in the field of observing cultures of cells, such as medicine, biology, pharmacy, and agriculture.
１ 筐体
２ 細胞培養庫
３ 透過照明光学系
３ａ 落射照明光学系
３ａ1 明るさ絞り
３ａ2 レンズ
３ａ3 ハーフミラー
３ｂ 対物レンズ
３ｃ コンデンサレンズ
３ｄ 反射部材
４ 結像光学系
４ａ 偏向ミラー
４ｂ 結像レンズ
５ 照明用光源
６ 撮像素子
７ 画像処理装置
８，８’ リングスリット
９ 位相板
１０ 培養標本（試料）
１１1，１１2，・・・１１n シャーレ
１２ 移動ステージ
１３ λ／４板
１４ 偏光子
１５ 検光子
１６ リレー光学系
１６ａ，１６ｂ レンズ
DESCRIPTION OF SYMBOLS 1 Case 2 Cell culture chamber 3 Transmission illumination optical system 3a Epi-illumination optical system 3a 1 Brightness stop 3a 2 Lens 3a 3 Half mirror 3b Objective lens 3c Condenser lens 3d Reflective member 4 Imaging optical system 4a Deflection mirror 4b Imaging lens DESCRIPTION OF SYMBOLS 5 Illumination light source 6 Image sensor 7 Image processing apparatus 8,8 'Ring slit 9 Phase plate 10 Culture specimen (sample)
11 1 , 11 2 ,... 11 n Petri dish 12 Moving stage 13 λ / 4 plate 14 Polarizer 15 Analyzer 16 Relay optical system 16a, 16b Lens
Claims (18)
筐体の内部に配置された、前記照明用光源からの光を試料側に導く落射照明光学系と、前記落射照明光学系からの光を試料上に集光する対物レンズと、前記試料を挟んで前記対物レンズと対向する位置に配置されたコンデンサレンズと、前記コンデンサレンズの前側焦点位置近傍に配置された反射部材とを備え、
前記反射部材からの反射光を用いて前記試料を透過照明するようにしたことを特徴とする透過照明装置。 A light source for illumination arranged outside the housing;
An epi-illumination optical system that guides light from the illumination light source to the sample side, an objective lens that condenses the light from the epi-illumination optical system on the sample, and the sample are disposed inside the housing And a condenser lens disposed at a position facing the objective lens, and a reflective member disposed near the front focal position of the condenser lens,
A transmission illumination apparatus, wherein the sample is transmitted and illuminated using reflected light from the reflection member.
前記対物レンズの瞳位置に、位相板を配置したことを特徴とする請求項１に記載の透過照明装置。 In the epi-illumination optical system, a ring slit is disposed at any one of a position conjugate with the pupil position of the objective lens and a front focal position of the condenser lens, and
The transmission illumination device according to claim 1, wherein a phase plate is disposed at a pupil position of the objective lens.
前記対物レンズの瞳位置に、位相板を配置したことを特徴とする請求項１に記載の透過照明装置。 A ring slit is disposed in both the position that is conjugate with the pupil position of the objective lens and the front focal position of the condenser lens in the incident light optical system,
The transmission illumination device according to claim 1, wherein a phase plate is disposed at a pupil position of the objective lens.
前記対物レンズの瞳位置と共役となる位置に、位相板を配置したことを特徴とする請求項１に記載の透過照明装置。 In the epi-illumination optical system, a ring slit is disposed at any one of a position conjugate with the pupil position of the objective lens and a front focal position of the condenser lens,
The transmitted illumination apparatus according to claim 1, wherein a phase plate is disposed at a position conjugate with a pupil position of the objective lens.
前記対物レンズの瞳位置と共役となる位置に、位相板を配置したことを特徴とする請求項１に記載の透過照明装置。 A ring slit is disposed in both the position that is conjugate with the pupil position of the objective lens and the front focal position of the condenser lens in the incident light optical system,
The transmitted illumination apparatus according to claim 1, wherein a phase plate is disposed at a position conjugate with a pupil position of the objective lens.
落射照明光の光路上に、偏光子を配置し、
透過照明光の光路上に、検光子を配置したことを特徴とする請求項１〜５のいずれかに記載の透過照明装置。 A λ / 4 plate is disposed between the sample and the reflecting member,
Place a polarizer on the optical path of epi-illumination light,
The transmitted illumination device according to claim 1, wherein an analyzer is disposed on an optical path of the transmitted illumination light.
前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、
前記照明位置に移動した試料から前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されていることを特徴とする請求項１〜６のいずれかに記載の透過照明装置。 A cell culture chamber for culturing a plurality of samples inside the housing,
The cell culture chamber includes a moving unit capable of moving a predetermined sample to an illumination position among the plurality of samples,
The optical member arrange | positioned on the optical path from the sample which moved to the said illumination position to the said reflection member is arrange | positioned inside the said cell culture box, The any one of Claims 1-6 characterized by the above-mentioned. Transmission lighting device.
前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、
前記位相板から前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されていることを特徴とする請求項２又は３に記載の透過照明装置。 A cell culture chamber for culturing a plurality of samples inside the housing,
The cell culture chamber includes a moving unit capable of moving a predetermined sample to an illumination position among the plurality of samples,
The transmission illumination device according to claim 2 or 3, wherein an optical member disposed on an optical path from the phase plate to the reflection member is disposed inside the cell culture box.
前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、
前記対物レンズから前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されていることを特徴とする請求項４又は５に記載の透過照明装置。 A cell culture chamber for culturing a plurality of samples inside the housing,
The cell culture chamber includes a moving unit capable of moving a predetermined sample to an illumination position among the plurality of samples,
6. The transmission illumination device according to claim 4, wherein an optical member disposed on an optical path from the objective lens to the reflecting member is disposed inside the cell culture box.
前記筐体の内部に、前記透過照明装置を介して透過照明された前記試料の像を観察する観察光学系を備えた顕微鏡。 The transmission illumination device according to any one of claims 1 to 9,
A microscope provided with an observation optical system for observing an image of the sample that is transmitted and illuminated through the transmission illumination device inside the housing.
前記筐体の内部に、前記透過観察装置を介して透過照明された前記試料の像を観察する観察光学系を備え、
前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置のいずれかに、リングスリットを配置するとともに、
前記観察光学系内において前記対物レンズの瞳位置と共役となる位置に、位相板を配置したことを特徴とする顕微鏡。 An illumination light source disposed outside the housing; an epi-illumination optical system disposed inside the housing for guiding light from the illumination light source toward the sample; and the light from the epi-illumination optical system as a sample An objective lens that condenses on the top, a condenser lens that is disposed at a position facing the objective lens across the sample, and a reflective member that is disposed near a front focal position of the condenser lens, the reflective member A transmission illumination device adapted to transmit and illuminate the sample using reflected light from
An observation optical system for observing an image of the sample that is transmitted and illuminated through the transmission observation device is provided inside the housing.
In the epi-illumination optical system, a ring slit is disposed at any one of a position conjugate with the pupil position of the objective lens and a front focal position of the condenser lens,
A microscope characterized in that a phase plate is arranged at a position conjugate with the pupil position of the objective lens in the observation optical system.
前記筐体の内部に、前記透過観察装置を介して透過照明された前記試料の像を観察する観察光学系を備え、
前記落射光学系内において前記対物レンズの瞳位置と共役となる位置、及び前記コンデンサレンズの前側焦点位置の両方に、リングスリットを配置するとともに、
前記観察光学系内において前記対物レンズの瞳位置と共役となる位置に、位相板を配置したことを特徴とする顕微鏡。 An illumination light source disposed outside the housing; an epi-illumination optical system disposed inside the housing for guiding light from the illumination light source toward the sample; and the light from the epi-illumination optical system as a sample An objective lens that condenses on the top, a condenser lens that is disposed at a position facing the objective lens across the sample, and a reflective member that is disposed near a front focal position of the condenser lens, the reflective member A transmission illumination device adapted to transmit and illuminate the sample using reflected light from
An observation optical system for observing an image of the sample that is transmitted and illuminated through the transmission observation device is provided inside the housing.
A ring slit is disposed in both the position that is conjugate with the pupil position of the objective lens and the front focal position of the condenser lens in the incident light optical system,
A microscope characterized in that a phase plate is arranged at a position conjugate with the pupil position of the objective lens in the observation optical system.
落射照明光の光路上に、偏光子を配置し、
透過照明光の光路上に、検光子を配置したことを特徴とする請求項１２又は１３に記載の顕微鏡。 A λ / 4 plate is disposed between the sample and the reflecting member,
Place a polarizer on the optical path of epi-illumination light,
The microscope according to claim 12 or 13, wherein an analyzer is disposed on the optical path of the transmitted illumination light.
前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、
前記照明位置に移動した試料から前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されていることを特徴とする請求項１２〜１４のいずれかに記載の顕微鏡。 A cell culture chamber for culturing a plurality of samples inside the housing,
The cell culture chamber includes a moving unit capable of moving a predetermined sample to an illumination position among the plurality of samples,
The optical member arranged on the optical path from the sample moved to the illumination position to the reflecting member is arranged inside the cell culture box. Microscope.
前記細胞培養庫が、前記複数の試料のうち、所定の試料を照明位置に移動可能な移動手段を備え、
前記対物レンズから前記反射部材までの光路上に配置される光学部材が、前記前記細胞培養庫の内部に配置されていることを特徴とする請求項１２〜１４のいずれかに記載の顕微鏡。 A cell culture chamber for culturing a plurality of samples inside the housing,
The cell culture chamber includes a moving unit capable of moving a predetermined sample to an illumination position among the plurality of samples,
The microscope according to any one of claims 12 to 14, wherein an optical member arranged on an optical path from the objective lens to the reflecting member is arranged inside the cell culture box.
落射照明したときに前記試料を透過した落射光を反射部材で反射し、
反射した光で前記試料を透過照明することを特徴とする透過照明方法。 Using the light from the light source for illumination arranged outside the housing, the sample placed inside the housing is incidentally illuminated,
Reflecting the reflected light that has passed through the sample when reflected on the reflecting member,
A transmission illumination method, wherein the sample is transmitted and illuminated with reflected light.
落射照明したときに前記試料を透過した落射光を前記反射部材で反射し、
反射した光で前記試料を透過照明することを特徴とする透過照明方法。 Through the transmission illumination device according to any one of claims 7 to 9, the sample in the cell culture box is incidentally illuminated,
Reflecting the epi-illuminated light that has passed through the sample when the epi-illuminator is illuminated,
A transmission illumination method, wherein the sample is transmitted and illuminated with reflected light.
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