JP7031740B2 - Phase plate, objective lens, and observation device - Google Patents

Description

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

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 phase difference of the resulting light into contrast. By doing so, the light and dark images of the subject are observed. A 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 by 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 the subject with a brighter contrast than the background requires a phase plate different from the dark contrast method.
[Patent Document 1] Japanese Unexamined Patent Publication No. 6-289438

General disclosure

(Item 1)
The phase plate may include a translucent substrate.
The phase plate is a first material formed on a substrate from a first material having a higher refractive index as the wavelength is longer, which modulates the transmittance of light and delays the phase of transmitted light by a quarter wavelength with respect to the wavelength range. It may be provided with a modulator.
The phase plate may include a second modulation unit formed around the first modulation unit on the substrate to modulate the transmittance of light to a transmittance different from the transmittance of the first modulation unit.
(Item 2)
The wavelength range may be the wavelength range of visible light.
(Item 3)
The first material may contain at least one of titanium (Ti), chromium (Cr), and tantalum (Ta).
(Item 4)
The upper surface of the first modulation unit may be covered with a second material having a lower refractive index as the wavelength becomes longer.
(Item 5)
The second material may be an oxide of the first material.
(Item 6)
The second modulation section may be formed by using the first material and having a thickness different from that of the first modulation section.
(Item 7)
The second modulation section may have a thickness smaller than that of the first modulation section.
(Item 8)
The first modulation section and the second modulation section are the first portion formed in the region on the substrate occupied by the first modulation section and the second modulation section with a thickness equal to that of the second modulation section, and the first portion on the first portion. The region occupied by one modulation unit may have a second portion formed with a thickness equal to the difference in thickness between the first modulation unit and the second 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 device may include the objective lens according to item 10.

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

The outline of the above invention does not list all the features of the present invention. A subcombination of these feature groups can also be an invention.

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

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

1 and 2 show the 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 with respect to the reference line BB in FIG. The phase plate 1 is a phase plate that modulates the light transmittance and phase that can be used in phase contrast 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 is light obtained by diffracting the phase of direct light transmitted through a subject (that is, a medium and a phase object in the subject) by a phase object in the subject having a higher refractive index than the medium (that is, the phase object). This is a contrast method in which the subject is brightened and the background is darkened by delaying the phase difference between the direct light and the diffracted light by 1/4 wavelength with respect to the diffracted light.

The substrate 2 is a plate-shaped member that supports the first and second modulation units 3a and 4a. As the substrate 2, a member having transparency in the visible light region (for example, a wavelength region of 380 to 780 nm), for example, a glass substrate can be adopted. The upper surface of the substrate 2 on which the first and second modulation units 3a and 4a, which will be described later, are not formed, that is, the exposed surfaces inside and outside the ring-shaped first and second modulation units 3a and 4a also have light transmittance. It forms a non-modulation section 2a that does not modulate the phase.

The first modulation unit 3a is a film body that modulates the transmittance of light and delays the phase of the transmitted light by a quarter wavelength with respect to the light passing through an air layer having the same thickness. The first modulation unit 3a is formed on the substrate 2 in an annular shape corresponding to the case where the subject is illuminated and observed by using the annular illumination as an example. It should be noted that the shape is not limited to the ring band, and may be any narrow shape such as a circle or a rectangle corresponding to the lighting shape. The first modulation unit 3a includes a main body 3b and an adjustment film 3c.

The main body 3b is a portion formed directly above the substrate 2. The main body 3b is a material having a light transmission rate of less than 1 and a refractive index of 1 or more in a visible light region (meaning a wavelength range of visible light, particularly a reference wavelength of 550 nm), and a material having a higher refractive index as the wavelength is longer. Can be formed from. Such a wavelength characteristic of the refractive index is also referred to as a dedispersible material, and a material having a dedispersible property is also referred to as a dedispersible material. A material may be used in which the phase delay of the transmitted light increases with increasing wavelength. As such a back-dispersible material, titanium (Ti), chromium (Cr), or tantalum (Ta) can be used. By using these metals, the light transmittance can be sufficiently reduced with a small film thickness, which makes it possible to clearly project the details of the subject in the phase contrast observation by the bright contrast method.

The adjusting film 3c is a portion that covers the upper surface of the main body 3b in order to adjust the wavelength dispersibility of the first modulation unit 3a by the main body 3b. The adjusting film 3c can be formed from a material having a lower refractive index as the wavelength becomes longer. Such a wavelength characteristic of the refractive index is also referred to as positive dispersibility, and a material having positive dispersibility is also referred to as a positive dispersibility material. A material may be used in which the phase delay of the transmitted light decreases with increasing wavelength. As a result, the phase wavelength dispersion is adjusted by the first modulation unit 3a, and the wavelength dispersion of the desired phase can be realized only by the two layers of the main body 3b and the adjustment film 3c. As such a positively dispersible material, an oxide of the material forming the main body 3b, for example, TiO ₂ , Cr ₂ O ₃ or Ta ₂ O ₅ can be used. By showing contradictory dispersibility between the material forming the main body 3b and the material forming the adjusting film 3c, in addition to being able to form the first modulation unit 3a using these, the same metal can be used for each. By including it, the same resistance to the chemicals and the like used in the manufacturing process of the phase plate 1 is exhibited, and the phase plate 1 can be easily manufactured.

The second modulation unit 4a is a film body that modulates the light transmittance to a transmittance different from that of the first modulation unit 3a (however, less than 1). The second modulation unit 4a is formed on the substrate 2 so as to sandwich the periphery of the first modulation unit 3a, that is, the ring-shaped first modulation unit 3a between two ring bands arranged inside and outside the ring band. ing. The second modulation unit 4a is formed by using the same material as the material used for forming the main body 3b of the first modulation unit 3a, but with a film thickness different from that of 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 film thickness smaller than that of the first modulation unit 3a. As a result, the transmittance of the second modulation unit 4a becomes larger than the transmittance of the first modulation unit 3a, and the phase modulation of the transmitted light becomes smaller.

A method of forming the first and second modulation units 3a and 4a on the substrate 2 will be described.

First, a first resist film having an annular region occupied by the first and second modulation units 3a and 4a is formed on the substrate 2.

Next, by a sputtering method, the first resist film is used as a mask, and the above-mentioned back-dispersible material is deposited on the substrate 2 to form a ring-shaped first layer 4. The thickness of the first layer 4 is determined to be a thickness at which the desired transmittance of the second modulation unit 4a can be obtained. For example, when tantalum (Ta) is used as the back-dispersible material, the thickness of the first layer 4 is about 25 nm. As a result, the transmittance of the second modulation unit 4a, about 8%, is obtained, and when observing a subject having a large phase difference, halo, that is, blurring of light in the shape of a shading that may occur around the object image occurs. No, good contrast can be obtained.

Next, a second resist film in which the annular region occupied by the first modulation unit 3a opens is formed on the first resist film on the substrate 2 or on the substrate 2 from which the first resist film has been peeled off. When peeling the resist film on the substrate 2, a peeling liquid may be used.

Next, by a sputtering method, a second resist film is used as a mask, and the same material as the previously used back-dispersible material is deposited on the first layer 4 to form a ring-shaped second layer 3. .. The thickness of the second layer 3 is determined to be a thickness at which the 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). As a result, the transmittance of the first modulation unit 3a (main body 3b) of about 2% can be obtained. Due to the small transmittance of the first modulation unit 3a, it is possible to detect a minute phase difference of the diffracted light, that is, the resolving power of the test object is increased.

By the above steps, as shown in FIG. 1, a ring-shaped main body 3b of the first modulation unit 3a and a second modulation unit 4a surrounding the main body 3b 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.

Further, by the sputtering method, the second resist film is used as a mask, and the positively dispersible material is deposited on the second layer 3 to form the adjusting film 3c. Here, as the positive dispersibility material, the oxide of the dedispersible material used above can be used. For example, when tantalum (Ta) is used as the dedispersible material, Ta ₂ O ₅ can be used as the positive dispersibility material. As a result, the wavelength dispersion of the phase by the first modulation unit 3a is adjusted, and the phase of the transmitted light in the visible light region is divided into 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 realized.

Finally, the phase plate 1 is obtained by peeling the resist film from the substrate 2. The resist film may be peeled off using a stripping solution. Here, the first and second modulation units 3a and 4a formed on the substrate 2 have a film thickness equal to that of the second modulation unit 4a in the region on the substrate 2 occupied by the first and second modulation units 3a and 4a. The second layer 3 formed in the region occupied by the first modulation section 3a on the molded first layer 4 and the first layer 4 with a film thickness equal to the difference in thickness between the first and second modulation sections 3a and 4a. Consists of including. With such a laminated structure, the first and second modulation units 3a and 4a can be easily aligned and formed on the substrate 2.

FIG. 3 shows an example (example) of the wavelength dispersion of the phase by the phase plate 1 configured as described above. Here, on the left and right of the drawing, the wavelength dispersion of the refractive index of the first modulation unit 3a and the wavelength dispersion of the phase of the transmitted light in the visible light region, respectively (the phase delay of the light transmitted through the first modulation unit 3a). Is shown. The dedispersible material titanium (Ti), chromium (Cr), or tantalum (Ta) is used to form the main body 3b, and the positive dispersible material, that is, the oxides of the material forming the main body 3b, TIO ₂ , Cr ₂ O. By forming the adjusting film 3c using ₃ or Ta ₂ O ₅ , 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 delayed by 90 degrees (based on 360 degrees) with respect to the wavelength in the visible light region.

FIG. 4 shows a comparative example of the wavelength dispersion of the phase by the phase plate. Here, on the left and right of the drawing, the wavelength dispersion of the refractive index of the first modulation unit 3a and the wavelength dispersion of the phase of the transmitted light in the visible light region, respectively (the phase delay of the light transmitted through the first modulation unit 3a). Is shown. When the refractive index of the first modulation unit 3a is almost 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 increasing wavelength in the visible light region. do.

FIG. 5 shows another comparative example of the wavelength dispersion of the phase by the phase plate. Here, on the left and right of the drawing, the wavelength dispersion of the refractive index of the first modulation unit 3a and the wavelength dispersion of the phase of the transmitted light in the visible light region, respectively (the phase delay of the light transmitted through the first modulation unit 3a). Is shown. When the refractive index of the first modulation unit 3a decreases with increasing wavelength as in this example, the phase delay of the light transmitted through the first modulation unit 3a is larger than that with respect to the increase in wavelength in the visible light region. Increase strongly.

As described in detail above, the phase plate 1 according to the present embodiment modulates the transmittance of light formed on the translucent substrate 2 and the substrate 2 from a material having a higher refractive index as the wavelength becomes longer. At the same time, the transmittance of light formed around the first modulation unit 3a that delays the phase of transmitted light by a quarter wavelength with respect to the wavelength range of visible light and the first modulation unit 3a on the substrate 2 is determined. A second modulation unit 4a that modulates the transmittance to a transmittance different from the transmittance of the first modulation unit 3a is provided. By forming the first modulation section 3a on the substrate 2 using a material having a higher refractive index as the wavelength is longer, the phase of the light transmitted through the first modulation section 3a for any wavelength in the visible light region can be adjusted. It is possible to realize a phase characteristic that delays the phase of light passing through an air layer of the same thickness by a quarter wavelength with respect to the phase of light passing through the unmodulated portion 2a, which is used in phase difference observation by the bright contrast method. It becomes possible to provide a possible phase plate.

FIG. 6 shows a schematic configuration of a phase contrast microscope 10 (an example of an observation device) using the phase plate 1 according to the present embodiment. The phase-contrast microscope 10 includes a light source LS, an aperture AP, a lens G1, and an objective lens G. The light source LS produces illumination light. The aperture AP has a ring-shaped opening and is arranged at the front focal position F of the lens G1. The lens G1 collects the illumination light through the aperture AP. The objective lens G has a pair of lenses G2 and G3 and a phase plate 1 arranged between them. 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 shape of the aperture of the aperture AP and the combined magnification of the lenses G1 and G2. 1 The modulation unit 3a is arranged at a position conjugate with the aperture of the aperture AP.

In the phase-contrast microscope 10 having the above configuration, the illumination light emitted from the light source LS is restricted in a ring shape by passing through the diaphragm AP, and is condensed by the lens G1 to illuminate the subject O. The light transmitted through the subject O is focused on the image plane 11 by the objective lens G and imaged. Here, the illumination light that illuminates the subject O is divided into direct light L1 transmitted through the subject and diffracted light (± primary) L2 diffracted by the subject, and the first modulation of the phase plate 1 is performed, respectively. It passes through the unit 3a and the non-modulation unit 2a. The angle difference between the direct light L1 and each of the ± 1st-order diffracted light L2 is defined as the diffraction angle θ. With 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 focused on the image plane 11 and interfere with each other, so that the phase difference between them is reproduced as the brightness and darkness of the image, and the subject O is displayed. Can be observed.

When observing a subject having a large structure (phase difference amount), the diffraction angle θ becomes small and the intensity of the diffracted light L2 becomes large. Therefore, the separation distance between the direct light L1 and the diffracted light L2 becomes small, and the direct light L1 passes through the first modulation unit 3a and the diffracted light L2 passes through the second modulation unit 4a. Therefore, the ratio of the transmittances of the first and second modulation units 3a and 4a is substantially the transmittance modulation of the direct light L1 with respect to the diffracted light L2, so that a low-contrast observation image can be obtained.

Further, when observing an object having a small structure (phase difference amount), the diffraction angle θ becomes large and the intensity of the diffracted light L2 becomes small. Therefore, the separation distance between the direct light L1 and the diffracted light L2 becomes large, and the direct light L1 passes through the first modulation section 3a and the diffracted light L2 passes through the non-modulation section 2a. Therefore, the ratio of the transmittances 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, so that only the amplitude of the direct light L1 is reduced. Observation image of can be obtained.

By using the phase plate 1, the transmittance is gradually different between the first modulation unit 3a, the second modulation unit 4a, and the non-modulation unit 2a, so that the light L1 and the direct light are diffracted regardless of the structure of the test object. Since the amplitude (light amount ratio) with the light L2 is appropriately modulated, preferably substantially equal to each other, it is possible to obtain a high-contrast observation image that brightly reflects the subject on a dark background in the bright contrast method. ..

FIG. 7 shows an example and a comparative example of a light-dark image obtained by observing a subject with a phase-contrast microscope 10 using the phase plate 1 according to the present embodiment. (A) to (C) are a dark contrast method with low contrast using the objective lens DLL, a dark contrast method with relatively high contrast using the objective lens DM, and a relatively high contrast method using the objective lens BM, respectively. It is an image obtained by the high bright contrast method. (D) is an image obtained by a high-contrast bright contrast method (appointment bright contrast method) using an objective lens ABH by a phase-contrast microscope 10 provided with a phase plate 1 according to the present embodiment. As a subject, an early mouse embryo (size: about 80 μm in diameter) was used. In the images (A) and (B), the inside of the subject is projected darkly against a bright background. In the image (C), the inside of the subject is brightly projected against a dark background. However, these have halos. 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 projected with a tomographic bright contrast without halo.

In the phase plate 1 according to the present embodiment, titanium (Ti), chromium (Cr), or tantalum (Ta) is used as the material for forming the main body 3b of the first modulation unit 3a. However, any combination thereof may be used, and other materials may be contained. When a plurality of materials are used, one film body containing them may be formed on the substrate 2, or a plurality of layers including each may be laminated on the substrate 2.

In the phase plate 1 according to the present embodiment, the second modulation unit 4a is formed by using the same material as the main body 3b of the first modulation unit 3a, but may be formed by using a different material. In such cases, for example, titanium (Ti), chromium (Cr), nickel (Ni), niobium (Nb), silver (Ag), tantalum (Ta), gold (Au), and inconel (chromium, iron, silicon). One of the nickel alloys including, etc.) or any combination may be used.

The phase-contrast microscope 10 according to the present embodiment is configured to include an objective lens G having a phase plate 1, but the present invention is not limited to this, and the phase-contrast microscope 1 and the objective lens G are separately included. It may be configured. In such a case, the phase plate 1 and the exit pupil surface of the objective lens G are arranged at positions optically conjugate to each other. Thereby, by the bright contrast method, a minute subject can be brightly projected and observed on a dark background.

Although the present invention has been described above using the embodiments, the technical scope of the present invention is not limited to the scope described in the above embodiments. It will be apparent to those skilled in the art that various changes or improvements can be made to the above embodiments. It is clear from the claims that embodiments with such modifications or improvements may also be included in the technical scope of the invention.

The order of execution of operations, procedures, steps, steps, etc. in the equipment, system, program, and method shown in the claims, description, and drawings is particularly "before" and "prior". It should be noted that it can be realized in any order unless the output of the previous process is used in the subsequent process. Even if the claims, the description, and the operation flow in the drawings are explained using "first", "next", etc. for convenience, it means that it is essential to carry out in this order. is not it.

1 ... phase plate, 2 ... substrate, 2a ... non-modulation section, 3 ... second layer, 3a ... first modulation section, 3b ... main body, 3c ... adjustment film, 4 ... first layer, 4a ... second modulation section, 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 ... Subject.

Claims (8)

With a translucent substrate,
A first modulator, which is formed on the substrate and has a characteristic that the longer the wavelength is, the higher the refractive index is, and delays the phase of transmitted light by a quarter wavelength with respect to the wavelength range of visible light.
A second modulation section formed around the first modulation section on the substrate and having a higher transmittance than the first modulation section,
In a region other than the first modulation section and the second modulation section, a non-modulation section having a higher transmittance than the second modulation section and a non-modulation section.
Equipped with
The first modulation unit has a main body portion formed of a first material having a higher refractive index as the wavelength is longer, and an adjusting film formed of a second material having a lower refractive index as the wavelength is longer.
The second modulation unit is thinner than the main body portion of the first modulation unit and is formed of the same first material as the main body portion of the first modulation unit.
Phase plate. The phase plate according to claim 1, wherein the first material contains at least one of titanium (Ti), chromium (Cr), and tantalum (Ta). The phase plate according to claim 1 or 2 , wherein the second material is an oxide of the first material. The second material is titanium oxide (TIO). ₂ ), Chromium oxide (Cr ₂ O ₃ ), Tantalum pentoxide (Ta ₂ O ₅ ), The phase plate according to claim 3. The first modulation section has a ring band shape, the second modulation section has a ring band shape adjacent to the inside and the outside of the first modulation section, and the non-modulation section has a second inside. The phase plate according to any one of claims 1 to 4 , which is a circular region inside the modulation unit and a region outside the second modulation unit outside. An objective lens comprising the phase plate according to any one of claims 1 to 5 . An observation device including the objective lens according to claim 6 . The phase plate according to any one of claims 1 to 5 .
An objective lens arranged at a position conjugate with the phase plate,
An observation device equipped with.

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