WO2019235511A1

WO2019235511A1 - Phase plate, objective lens, and observation device

Info

Publication number: WO2019235511A1
Application number: PCT/JP2019/022287
Authority: WO
Inventors: 大瀧　達朗; 隆一星加
Original assignee: 株式会社ニコン
Priority date: 2018-06-05
Filing date: 2019-06-05
Publication date: 2019-12-12
Also published as: JPWO2019235511A1; JP7031740B2

Abstract

A phase plate 1 according to an embodiment of the present invention is provided with: a translucent base plate 2; a first modulating portion 3a which is formed on the base plate from a material having a refractive index that is higher the longer the wavelength, and which modulates the transmittance of light and delays the phase of transmitted light by a quarter of a wavelength with respect to the wavelength region of visible light; and a second modulating portion 4a which is formed on the base plate around the first modulating portion, and which modulates the transmittance of light to a transmittance different from the transmittance in the first modulating portion. Forming the first modulating portion on the base plate using a material having a refractive index that is higher the longer the wavelength makes it possible to achieve a phase characteristic whereby, for an arbitrarily defined wavelength in the visible light region, the phase of light transmitted through the first modulating portion is delayed by a quarter of a wavelength relative to the phase of light passing through an air layer having the same thickness, thereby making it possible to provide a phase plate that can be used in phase difference observation employing a bright contrast method.

Description

Phase plate, objective lens, and observation device

The present invention relates to a phase plate, an objective lens, and an observation apparatus.

The phase contrast microscope illuminates the subject with illumination light, modulates the phase of the light diffracted by the subject through a phase plate arranged in the objective lens, and converts the resulting phase difference of light into contrast By doing so, the bright and dark image of the subject is observed. The phase contrast microscope can observe a colorless and transparent subject such as a biological specimen without staining. For example, Patent Document 1 discloses a phase plate that can be used in a phase contrast microscope using a dark contrast method for observing a subject (a phase object having a refractive index higher than that of a medium) with a darker contrast than the background. The bright contrast method for observing a subject with a brighter contrast than the background requires a phase plate different from the dark contrast method.
[Patent Document 1] Japanese Patent Laid-Open No. 6-289438

General disclosure

(Item 1)
The phase plate may include a translucent substrate.
The phase plate is formed of a first material having a higher refractive index as the wavelength is longer on the substrate. The phase plate modulates the light transmittance and delays the phase of the transmitted light by a quarter wavelength with respect to the wavelength region. A modulation unit may be provided.
The phase plate may include a second modulation unit that is formed around the first modulation unit on the substrate and modulates the light transmittance to a transmittance different from the transmittance of the first modulation unit.
(Item 2)
The wavelength range may be a visible wavelength range.
(Item 3)
The first material may include at least one of titanium (Ti), chromium (Cr), and tantalum (Ta).
(Item 4)
The upper surface of the first modulator may be covered with a second material having a lower refractive index as the wavelength is longer.
(Item 5)
The second material may be an oxide of the first material.
(Item 6)
The second modulation unit may be formed with a thickness different from that of the first modulation unit using the first material.
(Item 7)
The second modulation unit may have a smaller thickness than the first modulation unit.
(Item 8)
The first modulation unit and the second modulation unit include a first portion formed in a region on the substrate occupied by the first modulation unit and the second modulation unit with a thickness equal to the second modulation unit, and a first portion on the first portion. And a second portion formed with a thickness equal to a difference between the thicknesses of the first modulation unit and the second modulation unit in a region occupied by one modulation unit.
(Item 9)
The first modulation unit may have an annular shape.

(Item 10)
The objective lens may include the phase plate according to any one of items 1 to 9.

(Item 11)
The observation apparatus may include the objective lens described in Item 10.

The observation apparatus may include the phase plate according to any one of items 1 to 9 and an objective lens disposed at a position conjugate with the phase plate.

Note that the above summary of the invention does not enumerate all the features of the present invention. In addition, a sub-combination of these feature groups can also be an invention.

The structure of the phase plate which concerns on this embodiment is shown by a front view. The cross-sectional structure of the phase plate regarding the reference line BB in FIG. 1 is shown. An example of chromatic dispersion of a phase by a phase plate is shown. A comparative example of phase chromatic dispersion by a phase plate is shown. Another comparative example of phase chromatic dispersion by a phase plate is shown. 1 shows a schematic configuration of a phase contrast microscope. Examples and comparative examples of bright and dark images obtained by observing a subject with a phase contrast microscope using the phase plate according to the present embodiment are shown.

Hereinafter, the present invention will be described through embodiments of the invention. However, the following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.

1 and 2 show a configuration of the phase plate 1 according to the present embodiment. Here, FIG. 1 is a front view, and FIG. 2 is a cross-sectional view of the reference line BB in FIG. The phase plate 1 is a phase plate that modulates the transmittance and phase of light that can be used in phase difference observation by the bright contrast method, and includes a substrate 2, a first modulation unit 3a, and a second modulation unit 4a. The bright contrast method refers to light diffracted by a phase object in a subject having a higher refractive index than that of the medium (that is, a medium and a phase object in the subject). , Diffracted light) is a contrast method in which the phase difference between direct light and diffracted light is set to zero by delaying the wavelength by 1/4 wavelength to make the subject bright and the background dark.

The substrate 2 is a plate-like member that supports the first and

second modulators

3a and 4a. As the substrate 2, a member having translucency in the visible light region (for example, a wavelength region of 380 to 780 nm), for example, a glass substrate can be adopted. Note that the upper surface of the substrate 2 where the first and

second modulation sections

3a and 4a, which will be described later, are not formed, that is, the inner and outer exposed surfaces of the ring-shaped first and

second modulation sections

3a and 4a also have light transmittance. The non-modulation part 2a which does not modulate a phase is made.

The first modulator 3a is a film body that modulates the light transmittance and delays the phase of the transmitted light by a quarter wavelength with respect to the light passing through the air layer of the same thickness. The first modulation unit 3a is formed in an annular shape on the substrate 2 corresponding to the case where the object to be observed is observed using annular illumination as an example. In addition, it is not limited to the annular zone, and may have an arbitrary small shape such as a circle or a rectangle corresponding to the illumination shape. The first modulation unit 3a includes a main body 3b and an adjustment film 3c.

The main body 3b is a portion formed immediately above the substrate 2. The main body 3b is a material having a light transmittance of less than 1 and a refractive index of 1 or more in the visible light region (meaning the wavelength region of visible light, particularly the reference wavelength 550 nm), and the higher the wavelength, the higher the refractive index. Can be formed from Such a wavelength characteristic of the refractive index is also referred to as reverse dispersion, and a material having reverse dispersion is also referred to as reverse dispersion material. Note that a material in which the phase delay of transmitted light increases with respect to the increase in wavelength may be used. As such a reverse dispersion material, titanium (Ti), chromium (Cr), or tantalum (Ta) can be used. By using these metals, it is possible to sufficiently reduce the light transmittance with a small film thickness, so that the details of the test object can be clearly displayed in the phase difference observation by the bright contrast method.

The adjustment film 3c is a part that covers the upper surface of the main body 3b in order to adjust the wavelength dispersion of the first modulation unit 3a by the main body 3b. The adjustment film 3c can be formed of a material having a lower refractive index as the wavelength is longer. Such wavelength characteristics of refractive index are also referred to as positive dispersion, and a material having positive dispersion is also referred to as a positive dispersion material. Note that a material in which the phase delay of transmitted light decreases with an increase in wavelength may be used. Thereby, the chromatic dispersion of the phase by the first modulator 3a is adjusted, and the chromatic dispersion of the target phase can be realized by only two layers of the main body 3b and the adjustment film 3c. As such a positively dispersible material, an oxide of a material forming the main body 3b, for example, TiO ₂ , Cr ₂ O ₃ , or Ta ₂ O ₅ can be used. Since the material forming the main body 3b and the material forming the adjustment film 3c exhibit dispersibility opposite to each other, the first modulator 3a can be configured using these, and in addition, the same metal is used for each. By including, it shows the same tolerance with respect to the chemical | medical solution etc. which are used in the manufacturing process of the phase plate 1, and it becomes possible to manufacture the phase plate 1 easily.

The second modulation unit 4a is a film body that modulates the light transmittance to a different transmittance (but less than 1) from the first modulation unit 3a. The second modulation unit 4a is formed on the substrate 2 so as to sandwich the first modulation unit 3a around the first modulation unit 3a, that is, between the two annular zones arranged inside and outside the first modulation unit 3a. ing. The second modulation unit 4a is formed using the same material as that used to form the main body 3b of the first modulation unit 3a, but with a different film thickness from the first modulation unit 3a. That is, the light transmittance in the second modulation unit 4a is different from that in the first modulation unit 3a. In the present embodiment, the second modulation unit 4a is formed with a smaller film thickness than the first modulation unit 3a. As a result, the transmittance of the second modulator 4a is greater than the transmittance of the first modulator 3a, and the phase modulation of the transmitted light is reduced.

A method for forming the first and

second modulators

3a and 4a on the substrate 2 will be described.

First, a first resist film in which an annular region occupied by the first and

second modulators

3a and 4a is formed on the substrate 2 is formed.

Next, the first resist film is used as a mask by the sputtering method, and the above-mentioned reverse dispersive material is deposited on the substrate 2 to form the ring-shaped first layer 4. The thickness of the first layer 4 is determined so as to obtain a desired transmittance of the second modulator 4a. For example, when tantalum (Ta) is used as the reverse dispersion material, the thickness of the first layer 4 is about 25 nm. As a result, the transmittance of the second modulation unit 4a is about 8%, and when observing a test object having a large phase difference, a halo, that is, a blotting-like light blur that may occur around the object image occurs. And good contrast can be obtained.

Next, on the first resist film on the substrate 2 or on the substrate 2 from which the first resist film has been peeled off, a second resist film in which an annular region occupied by the first modulation section 3a is opened is formed. In addition, when peeling the resist film on the board | substrate 2, you may use stripping solution.

Next, by sputtering, the second resist film is used as a mask, and the same material as the previously used reverse dispersion material is deposited on the first layer 4 to form the annular zone-like second layer 3. . The thickness of the second layer 3 is determined such that a desired transmittance of the first modulation unit 3a can be obtained. For example, when tantalum (Ta) is used, the thickness of the second layer 3 is about 25 nm (the total thickness of the first and

second layers

4 and 3 is about 50 nm). Thereby, the transmittance of the first modulator 3a (main body 3b), approximately 2%, is obtained. The small transmittance of the first modulation unit 3a makes it possible to detect a minute phase difference of the diffracted light, that is, the resolution of the test object is increased.

Through the above-described steps, the main body 3b of the ring-shaped first modulation unit 3a and the second modulation unit 4a surrounding it are formed on the substrate 2 as shown in FIG. Therefore, it is confirmed that the product of the refractive index and the film thickness of the main body 3b of the first modulation unit 3a is approximately equal to a quarter of the wavelength of light in the entire visible light region.

Furthermore, by using the second resist film as a mask by sputtering, the above-described positively dispersible material is deposited on the second layer 3 to form the adjustment film 3c. Here, the oxide of the reverse dispersion material used previously can be used as the normal dispersion material. For example, when tantalum (Ta) is used as the reverse dispersion material, Ta ₂ O ₅ can be used as the normal dispersion material. Thereby, the wavelength dispersion of the phase by the first modulation unit 3a is adjusted, and in the visible light region, the phase of the transmitted light is four minutes with respect to the light passing through the air layer having the same thickness (that is, the light passing through the non-modulation unit 2a). A phase characteristic delayed by one wavelength is implemented.

Finally, the phase plate 1 is obtained by removing the resist film from the substrate 2. Note that the resist film may be stripped using a stripping solution. Here, the first and

second modulators

3a and 4a formed on the substrate 2 have a film thickness equal to that of the second modulator 4a in a region on the substrate 2 occupied by the first and

second modulators

3a and 4a. The second layer 3 formed with a film thickness equal to the difference between the thicknesses of the first and

second modulators

3a and 4a in the molded first layer 4 and the region occupied by the first modulator 3a on the first layer 4 It is comprised including. With such a laminated structure, the first and

second modulation sections

3a and 4a can be easily aligned and formed on the substrate 2.

FIG. 3 shows an example (example) of phase chromatic dispersion by the phase plate 1 configured as described above. Here, on the left and right of the drawing, respectively, the chromatic dispersion of the refractive index of the first modulation unit 3a and the chromatic dispersion of the phase of the transmitted light in the visible light region (the phase delay of the light transmitted through the first modulation unit 3a), respectively. Indicates. The inversely dispersible material titanium (Ti), chromium (Cr), or tantalum (Ta) is used to form the main body 3b, and the positively dispersive material, that is, the oxides of the material forming the main body 3b, TiO ₂ and Cr ₂ O ₃ or Ta ₂ O ₅ is used to form the adjustment film 3c, whereby the refractive index of the first modulation unit 3a increases in proportion to the wavelength. As a result, the phase of the light transmitted through the first modulation unit 3a is approximately 90 degrees behind the wavelength in the visible light region (based on 360 degrees) and delayed by 90 degrees.

FIG. 4 shows a comparative example of phase wavelength dispersion by the phase plate. Here, on the left and right of the drawing, respectively, the chromatic dispersion of the refractive index of the first modulator 3a and the chromatic dispersion of the phase of the transmitted light in the visible light region (the phase delay of the light transmitted through the first modulator 3a), respectively. Indicates. When the refractive index of the first modulation unit 3a is substantially constant with respect to the wavelength as in this example, the phase delay of the light transmitted through the first modulation unit 3a increases with an increase in wavelength in the visible light region. To do.

FIG. 5 shows another comparative example of phase wavelength dispersion by the phase plate. Here, on the left and right of the drawing, respectively, the chromatic dispersion of the refractive index of the first modulator 3a and the chromatic dispersion of the phase of the transmitted light in the visible light region (the phase delay of the light transmitted through the first modulator 3a), respectively. Indicates. When the refractive index of the first modulation unit 3a decreases as the wavelength increases as in this example, the phase delay of the light transmitted through the first modulation unit 3a is greater than the increase in wavelength in the visible light region. Strongly increases.

As described above in detail, the phase plate 1 according to the present embodiment modulates the light transmittance, which is formed on the translucent substrate 2 and the substrate 2 from a material having a higher refractive index as the wavelength is longer. Along with the first modulator 3a that delays the phase of the transmitted light by a quarter wavelength with respect to the wavelength range of visible light, and the light transmittance formed around the first modulator 3a on the substrate 2, A second modulator 4a that modulates the transmittance different from the transmittance of the first modulator 3a is provided. By forming the first modulation unit 3a on the substrate 2 using a material having a higher refractive index as the wavelength is longer, the phase of light transmitted through the first modulation unit 3a with respect to an arbitrary wavelength in the visible light region is changed. A phase characteristic can be realized that delays the wavelength by a quarter of the phase of light that passes through the air layer of the same thickness (that is, light that passes through the non-modulation part 2a), and is used for phase contrast observation by the bright contrast method. A possible phase plate can be provided.

FIG. 6 shows a schematic configuration of a phase contrast microscope 10 (an example of an observation apparatus) using the phase plate 1 according to the present embodiment. The phase contrast microscope 10 includes a light source LS, a diaphragm AP, a lens G1, and an objective lens G. The light source LS generates illumination light. The aperture AP has a ring-shaped opening and is disposed at the front focal position F of the lens G1. The lens G1 condenses illumination light through the aperture AP. The objective lens G includes a pair of lenses G2 and G3 and a phase plate 1 disposed therebetween. The phase plate 1 is conjugate with the aperture AP at the rear focal position F ′ of the lens G2, that is, the shape of the first modulation unit 3a is similar to the aperture shape of the aperture AP and the combined magnification of the lenses G1 and G2. One modulator 3a is arranged at a position conjugate with the aperture of the aperture AP.

In the phase contrast microscope 10 having the above-described configuration, the illumination light emitted from the light source LS is limited to a ring shape through the aperture AP, and is condensed by the lens G1 to illuminate the test object O. The light transmitted through the test object O is focused on the image plane 11 by the objective lens G and imaged. Here, the illumination light illuminating the test object O is divided into direct light L1 transmitted through the test object and diffracted light (± first order) L2 diffracted by the test object, and the first modulation of the phase plate 1 respectively. It passes through the part 3a and the non-modulation part 2a. Note that an angle difference between each of the direct light L1 and the ± first-order diffracted light L2 is a diffraction angle θ. Along with the diffraction, the phase of the direct light L1 is delayed by a quarter wavelength with respect to the diffracted light L2. Due to the action of the phase plate, the direct light L1 and the diffracted light L2 are collected on the image plane 11 and interfere with each other, so that the phase difference between them is reproduced as the brightness of the image, and the object O Can be observed.

When observing a test object having a large structure (phase difference amount), the diffraction angle θ decreases and the intensity of the diffracted light L2 increases. For this reason, the separation distance between the direct light L1 and the diffracted light L2 is reduced, and the direct light L1 is transmitted through the first modulating unit 3a and the diffracted light L2 is transmitted through the second modulating unit 4a. Therefore, the ratio of the respective transmittances of the first and

second modulators

3a and 4a becomes the substantial transmittance modulation of the direct light L1 with respect to the diffracted light L2, whereby a low-contrast observation image can be obtained.

Further, when observing a test object having a small structure (phase difference amount), the diffraction angle θ increases and the intensity of the diffracted light L2 decreases. For this reason, the separation distance between the direct light L1 and the diffracted light L2 is increased, and the direct light L1 is transmitted through the first modulating unit 3a and the diffracted light L2 is transmitted through the non-modulated unit 2a. Therefore, the ratio of the transmittance of each of the first modulation unit 3a and the non-modulation unit 2a is a substantial transmittance modulation of the direct light L1 with respect to the diffracted light L2, thereby reducing only the amplitude of the direct light L1. Can be obtained.

By using the phase plate 1, the transmittance varies stepwise between the first modulation unit 3a, the second modulation unit 4a, and the non-modulation unit 2a. Since the amplitude (light quantity ratio) with the light L2 is moderately modulated, and preferably modulated so as to be substantially equal, in the bright contrast method, it is possible to obtain an observation image with good contrast that reflects the subject brightly on a dark background. .

FIG. 7 shows examples and comparative examples of bright and dark images obtained by observing the subject with the phase-contrast microscope 10 using the phase plate 1 according to the present embodiment. (A) to (C) respectively show a dark contrast method with a low contrast using the objective lens DLL, a dark contrast method with a relatively high contrast using the objective lens DM, and a comparatively high contrast with the objective lens BM. It is an image obtained by a high bright contrast method. (D) is an image obtained by the bright contrast method (apodization bright contrast method) with high contrast using the objective lens ABH by the phase contrast microscope 10 including the phase plate 1 according to the present embodiment. In addition, the mouse | mouth early embryo (size is about 80 micrometers in diameter) was used as a test object. In the images (A) and (B), the inside of the subject is projected darkly against a bright background. In the image of (C), the inside of the subject is projected brightly against a dark background. However, these are hello. In the image of (D), the inside of the subject is bright against a dark background as in (C), but the inside of the subject is clearly displayed with a tomographically bright contrast without causing halo.

In the phase plate 1 according to this embodiment, titanium (Ti), chromium (Cr), or tantalum (Ta) is used as a material for forming the main body 3b of the first modulation unit 3a. Not limited thereto, any combination thereof may be used, and further other materials may be included. In the case of using a plurality of materials, one film body including them may be formed on the substrate 2, or a plurality of layers including each may be stacked on the substrate 2.

In the phase plate 1 according to this embodiment, the second modulation unit 4a is formed using the same material as that of the main body 3b of the first modulation unit 3a, but may be formed using a different material. In such a case, as an example, titanium (Ti), chromium (Cr), nickel (Ni), niobium (Nb), silver (Ag), tantalum (Ta), gold (Au), and inconel (chromium, iron, silicon) Or any combination thereof may be used.

The phase contrast microscope 10 according to the present embodiment is configured to include the objective lens G having the phase plate 1, but is not limited thereto, and includes the phase plate 1 and the objective lens G separately. It may be configured. In such a case, the phase plate 1 and the exit pupil plane of the objective lens G are disposed at optically conjugate positions. Thereby, a bright subject can be brightly viewed on a dark background by the bright contrast method.

As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

The execution order of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior”. It should be noted that they can be implemented in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for the sake of convenience, it means that it is essential to carry out in this order. is not.

DESCRIPTION OF SYMBOLS 1 ... Phase plate, 2 ... Board | substrate, 2a ... Non-modulation part, 3 ... 2nd layer, 3a ... 1st modulation part, 3b ... Main body, 3c ... Adjustment film, 4 ... 1st layer, 4a ... 2nd modulation part, DESCRIPTION OF SYMBOLS 10 ... Phase contrast microscope, 11 ... Image plane, AP ... Aperture, G ... Objective lens, G1, G2, G3 ... Lens, L1 ... Direct light, L2 ... Diffracted light, LS ... Light source, O ... Test object.

Claims

A translucent substrate;
A first modulation unit formed of a first material having a higher refractive index as the wavelength is longer on the substrate, which modulates the light transmittance and delays the phase of the transmitted light by a quarter wavelength with respect to the wavelength region; ,
A second modulation unit that is formed around the first modulation unit on the substrate and modulates the light transmittance to a transmittance different from the transmittance of the first modulation unit;
A phase plate.
The phase plate according to claim 1, wherein the wavelength range is a visible wavelength range.
The phase plate according to claim 1 or 2, wherein the first material includes at least one of titanium (Ti), chromium (Cr), and tantalum (Ta).
The phase plate according to any one of claims 1 to 3, wherein an upper surface of the first modulation section is covered with a second material having a lower refractive index as the wavelength is longer.
The phase plate according to claim 4, wherein the second material is an oxide of the first material.
The phase plate according to any one of claims 1 to 5, wherein the second modulation unit is formed with a thickness different from that of the first modulation unit using the first material.
The phase plate according to claim 6, wherein the second modulation section has a smaller thickness than the first modulation section.
The first modulation unit and the second modulation unit are formed in a region on the substrate occupied by the first modulation unit and the second modulation unit with a thickness equal to the second modulation unit, 8. A second portion formed in a region occupied by the first modulation portion on the first portion with a thickness equal to a difference in thickness between the first modulation portion and the second modulation portion. The phase plate described in 1.
The phase plate according to any one of claims 1 to 8, wherein the first modulation unit has an annular shape.
An objective lens comprising the phase plate according to any one of claims 1 to 9.
An observation apparatus comprising the objective lens according to claim 10.
The phase plate according to any one of claims 1 to 9,
An objective lens disposed at a position conjugate with the phase plate;
An observation apparatus comprising: