WO2024062667A1

WO2024062667A1 - Gradient light interference microscope

Info

Publication number: WO2024062667A1
Application number: PCT/JP2023/015723
Authority: WO
Inventors: 健一仁坂; 葵大西
Original assignee: 株式会社Ｓｃｒｅｅｎホールディングス
Priority date: 2022-09-21
Filing date: 2023-04-20
Publication date: 2024-03-28
Also published as: JP2024044416A

Abstract

This gradient light interference microscope (1) includes an incidence path (30) in which light is made incident from a light source (20) to a sample arrangement position (40), and an emission path (50) in which transmitted light or reflected light emitted from the sample arrangement position (40) is made incident on an imaging element (60). The incidence path (30) has a first polarizing double-image prism (32) that splits incident light into a first polarized wave and a second polarized wave orthogonal to each other, and a condenser lens (33), in the stated order. The emission path (50) has an objective lens (51), a second polarizing double-image prism (52), a branching mechanism (56) that branches a composite beam emitted from the second polarizing double-image prism (52) into a plurality of paths, a phase delay mechanism (58) that causes mutually different phase delays in each second polarized wave of the plurality of branched beams, and a polarizing plate (72), in the stated order. By branching a light wave and applying a phase delay to each branch, an image can be captured for a plurality of phase delay amounts simultaneously, and image capture time can be shortened.

Description

gradient light interference microscope

The present invention relates to an imaging device and an imaging method for performing quantitative phase imaging based on gradient light interference microscopy.

Phase contrast microscopes and differential interference microscopes have been developed and used as technologies for imaging colorless and transparent objects such as cells. The phase distribution obtained with these microscopes is a qualitative phase distribution, and quantitative information about cells cannot be obtained.

For example, in a differential interference microscope, the light that irradiates the sample is separated into P-polarized light and S-polarized light whose polarization directions are orthogonal to each other using a differential interference prism such as a Wollaston prism or Nomarski prism. Shift the irradiation position. The amount of deviation between the P polarized light and the S polarized light is referred to as the shear amount. Then, the transmitted light or reflected light from the sample of each polarized light wave is combined again by the prism to cause interference. The interference intensity at this time changes depending on the phase difference with respect to the shear amount. This makes it possible to visualize the amount of change (differentiation) in shape of colorless and transparent cells and uneven objects. However, although this method can visualize the amount of change in shape and phase, it cannot acquire quantitative information.

Therefore, in order to perform quantitative phase imaging that quantitatively acquires phase information of a measurement target, a gradient light called GLIM (Gradient Light Interference Microscopy) is used to acquire quantitative phase by adding a liquid crystal modulation element to a differential interference microscope. An interference microscopy method has been proposed. The gradient light interference microscopy method is described in, for example, Non-Patent Document 1.

In the gradient light interference microscopy method described in Non-Patent Document 1, a liquid crystal modulation element is used to add four different amounts of phase delay to one of the separated P-polarized light and S-polarized light whose irradiation positions are shifted. Then, the phase difference between the polarized lights is obtained from the interference intensity distribution in each of the four ways of multiplexing. Then, a local phase differential is calculated by dividing the phase difference between the polarized lights by the shear amount. Since the differential value of the phase is calculated at each position of the image, by integrating this, the phase distribution of the measurement target can be obtained.

However, in the gradient light interference microscopy method described in Non-Patent Document 1, in order to perform imaging for four different amounts of phase delay, imaging must be performed four times. That is, since the imaging time is four times longer than that of a conventional differential interference microscope, the application to dynamic samples is limited.

The present invention was made in view of these circumstances, and an object of the present invention is to provide a technique for shortening the imaging time in gradient light interference microscopy.

In order to solve the above problems, the first invention of the present application is a gradient light interference microscope that performs phase imaging using polarized waves, which includes an incident path for making light incident from a light source to a sample placement position, and a path from the sample placement position to the sample placement position. an output path that makes the emitted transmitted light or reflected light enter the imaging device, and the input path includes a first polarization double image that sequentially separates the incident light into orthogonal first polarized light waves and second polarized light waves. a prism; and a condenser lens disposed between the first polarizing double-image prism and the sample arrangement position, and the output path sequentially includes the transmitted light emitted from the sample arrangement position and the An objective lens into which the reflected light is incident, a second polarizing double-image prism that combines the first polarized light wave and the second polarized light wave, and a combined light emitted from the second polarized double-image prism into three paths. A branching mechanism that branches into the plurality of paths, a phase delay mechanism that causes different phase delays in the second polarized waves of each of the plurality of branched lights, and a phase delay mechanism that causes the first polarized waves of each of the plurality of branched lights; and a polarizing plate that aligns the polarization direction of the second polarized light wave.

The second invention of this application is the gradient light interference microscope of the first invention, in which the branching mechanism is a diffraction grating.

A third invention of the present application is the gradient light interference microscope according to the second invention, in which the branching mechanism is a phase grating.

A fourth invention of the present application is the gradient light interference microscope according to the third invention, in which the phase grating is provided so that a Fourier spectrum distribution appears two-dimensionally in two rows and two columns on the imaging surface of the imaging element.

A fifth invention of the present application is the gradient light interference microscope according to the fourth invention, wherein the phase delay mechanism is four wave plates arranged two-dimensionally in two rows and two columns, and the four wave plates are , the amount of phase delay is different from each other.

A sixth invention of the present application is the gradient light interference microscope according to any one of the first to fifth inventions, in which the phase delay mechanism is a plurality of wavelength plates corresponding to each of the branched lights.

A seventh invention of the present application is the gradient light interference microscope according to any one of the first to sixth inventions, wherein the branching mechanism branches the combined light into four or more paths, and the phase delay mechanism includes: A phase delay of 0, π/2, π, and 3π/2 is caused in the second polarized light wave of each of the four branched lights.

According to the first to seventh inventions of the present application, in the gradient light interference microscopy method, by splitting the separated first polarized light wave and the second polarized light wave and performing a phase delay on each, a plurality of phase delay amounts can be obtained. can be imaged simultaneously. Therefore, it is possible to shorten the imaging time in gradient light interference microscopy, which conventionally took a long time.

In particular, according to the second and third inventions of the present application, it is possible to branch light waves into a plurality of light waves without increasing the size of the device.

In particular, according to the fifth or sixth invention of the present application, a predetermined phase delay can be caused in only one of the two polarized waves for each of the plurality of branched lights without increasing the size of the device.

FIG. 1 is a schematic diagram of a gradient light interference microscope according to a first embodiment. FIG. 3 is a diagram showing the area distribution of the phase grating of the gradient light interference microscope according to the first embodiment. FIG. 3 is a diagram showing the Fourier spectrum distribution of the phase grating of the gradient light interference microscope according to the first embodiment. FIG. 3 is a diagram showing the arrangement of polarizing plates of the gradient light interference microscope according to the first embodiment. FIG. 1 is a schematic diagram showing how a reflected wave from a sample surface is observed using a polarizing birefringent prism. FIG. 3 is a schematic diagram of a gradient light interference microscope according to a second embodiment.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

<1. First embodiment>
Below, a microscope 1, which is a gradient light interference microscope according to an embodiment of the present invention, will be described with reference to FIG. FIG. 1 is a schematic diagram showing the configuration of a microscope 1. In order to image colorless and transparent objects such as cells, this microscope 1 uses gradient light interference microscopy to add multiple amounts of phase delay to one of two polarized waves whose irradiation position is shifted. The phase difference between polarized lights is obtained from the intensity distribution of the combination of two polarized lights.

As shown in FIG. 1, the microscope 1 includes a light source 20, an entrance path 30, a sample placement section 40, an exit path 50, an image sensor 60, and a calculation section 80. The microscope 1 of this embodiment irradiates light onto a sample placed in a sample placement section 40 and observes the transmitted light using an imaging device 60. For this reason, the input path 30 from the light source 20 to the sample placement section 40 and the output path 50 from the sample placement section 40 to the image sensor 60 are arranged in a straight line.

The light source 20 supplies light to irradiate the sample. The light emitted from the light source 20 is white light.

The incident path 30 is an optical system that allows light to enter the sample arrangement section 40 from the light source 20. The optical axes in the incident path 30 are arranged in a straight line. The incident path 30 includes a first polarizing plate 31, a first prism 32, and a condenser lens 33 in this order from the light source 20 toward the sample placement section 40.

The first polarizing plate 31 is an optical element that allows only light traveling in a specific direction to pass (transmit) among the light emitted from the light source 20.

The first prism 32 is a so-called differential interference prism (DIC prism) that separates incident light into two orthogonal polarized waves. The first prism 32 is a "first polarization double-image prism" that separates incident light into a first polarization wave and a second polarization wave that are perpendicular to each other. Specifically, the first prism 32 converts the incident light into a first polarized light wave having a vibration direction in a first direction perpendicular to the optical axis and a first polarized light wave having a vibration direction in a second direction perpendicular to the optical axis and the first direction. and the second polarized light wave having a predetermined separation angle. For example, a Wollaston prism or a Nomarski prism is used as the first prism 32.

The polarization axis (transmission axis) of the first polarizing plate 31 and the first direction and the second direction of the first prism 32 are arranged to intersect at an angle of 45°, respectively. Thereby, the optical energy of the first direction component and the second direction component of the polarized light wave emitted from the first polarizing plate 31 becomes equal. That is, the first polarized light wave and the second polarized light wave emitted from the first prism 32 have substantially the same intensity.

The condenser lens 33 is a lens for appropriately irradiating the first polarized light wave and the second polarized light wave separated by the first prism 32 onto the sample arrangement section 40. The focal plane P1 of the condenser lens 33 is arranged on a plane where the light transmitted through the first deflection plate 31 is separated into two polarized lights by the first prism 32 (that is, the localization plane of the first prism). Due to the condenser lens 33, the first polarized light wave and the second polarized light wave are shifted by a predetermined shear amount and travel in a direction perpendicular to the optical axis. The first polarized light wave and the second polarized light wave shifted by a predetermined shear amount are collectively referred to as "separation/combination."

The sample placement section 40 is a mechanism for placing a sample, which is a biological specimen, at the sample placement position P2. In practice, the sample placement section 40 is provided with a fixing mechanism for fixing a dish or a slide in which the sample is contained. The sample placed in the sample placement section 40 is irradiated with the separated and combined light emitted from the condenser lens 33.

The microscope 1 of this embodiment is for observing transmitted light irradiated onto a sample. Separated and multiplexed light incident on the sample from the light source 20 via the input path 30 passes through the sample and heads toward the output path 50 . At this time, the first polarized light wave and the second polarized light wave included in the separation/combination are transmitted through the sample at positions shifted by a predetermined shear amount. For this reason, the light transmitted through the sample becomes a separated and multiplexed wave in which the first polarized light wave and the second polarized light wave are shifted by the amount of shear.

The output path 50 is an optical system that allows the transmitted light from the sample placement section 40 to enter the image sensor 60. The exit path 50 includes an objective lens 51, a second prism 52, a color filter 53, a first imaging lens 54, a second imaging lens 55, a phase grating 56, and a second imaging lens 55 in order from the sample arrangement section 40 toward the imaging device 60. 1 lens 57 , a phase delay section 58 , a second lens 59 , a third lens 71 , and a second polarizing plate 72 .

The objective lens 51 is a lens that images the transmitted light (separated and combined waves including the separated first polarized light wave and the second polarized light wave) that has passed through the sample in the sample placement section 40.

The second prism 52 is arranged at the focal plane P3 of the objective lens 51. The second prism 52 is a differential interference prism (DIC prism) similar to the first prism. The second prism 52 is a "second polarized double-image prism" that combines the first polarized light wave and the second polarized light wave. Separated and combined waves in which the first polarized light wave and the second polarized light wave are shifted by a predetermined shear amount are incident on the second prism 52 . The second prism 52 eliminates the deviation between the first polarized light wave and the second polarized light wave by shifting the separated first polarized light wave and the second polarized light wave in the opposite direction to the first prism 32 (the amount of shear is reduced). 0), and outputs a composite wave of the first polarized light wave and the second polarized light wave. The second prism 52 is arranged on the focal plane P2 of the objective lens 51.

The color filter 53 passes only light in a predetermined wavelength range from the composite wave emitted from the second prism 52. When the light emitted from the light source 20 has a wide wavelength range, the separation angles at the first prism 32 and the second prism 52 differ depending on the wavelength, which may cause a difference in optical path length and cause the image to become blurred. By narrowing the wavelength range using the color filter 53, blurring of the image observed on the image sensor 60 can be suppressed.

The first imaging lens 54 and the second imaging lens 55 are lenses for forming an image of the composite wave emitted from the second prism 52 and passing through the color filter 53. The Fourier spectral distribution of the composite wave appears on the Fourier plane (pupil plane) P4 of the first imaging lens 54 and the second imaging lens 55.

The phase grating 56 is arranged on the Fourier plane (pupil plane) of the first imaging lens 54 and the second imaging lens 55. The phase grating 56 is a type of diffraction grating. The phase grating 56 of this embodiment is a transmission type phase grating that combines regions having different refractive indexes (modulating the phase). Note that the phase grating 56 may be formed by combining regions with different thicknesses.

FIG. 2 is a diagram showing the area distribution of the phase grating 56 of this embodiment. FIG. 3 is a diagram showing the point spread intensity distribution of the phase grating 56 of this embodiment. As shown in FIG. 2, this phase grating 56 has a checkerboard pattern in which regions in which no phase delay is performed (the amount of phase delay is 0) and regions in which the amount of phase delay is π are alternated two-dimensionally. It is arranged in a shape. The Fourier spectrum distribution of the phase grating 56 is a two-dimensional two-by-two distribution on the imaging plane of the second imaging lens 55 (or the imaging plane of the imaging element 60) as shown in FIG. The light waves from the sample appear convolved in the .

That is, the phase grating 56 is a branching mechanism that branches the composite wave emitted from the second prism 52 into three or more routes. The phase grating 56 of this embodiment separates the composite wave emitted from the second prism 52 and passed through the color filter 53 into four light waves arranged in two rows and two columns. Here, the four light waves separated by the phase grating 56 are respectively referred to as a first branch wave, a second branch wave, a third branch wave, and a fourth branch wave.

Each of the first branch wave, the second branch wave, the third branch wave, and the fourth branch wave includes a first polarized light wave having an amplitude in the first direction and a second polarized light wave having an amplitude in the second direction. include.

The first lens 57, the phase delay section 58, the second lens 59, the third lens 71, and the second polarizing plate 72 are arranged in this order between the phase grating 56 and the image sensor 60. Between the first lens 57 and the second lens 59, the first branched wave, the second branched wave, the third branched wave, and the fourth branched wave travel parallel to each other.

The phase delay unit 58 has a phase delay unit that causes different phase delays in second polarized waves included in each of the plurality of branched lights (a first branched wave, a second branched wave, a third branched wave, and a fourth branched wave). It is a delay mechanism. Note that the phase delay unit 58 applies a phase delay to the first polarized light included in each of the plurality of branched lights (first branched wave, second branched wave, third branched wave, and fourth branched wave). Don't let it happen.

As shown in FIG. 4, the phase delay unit 58 of this embodiment includes a frame 580, a first wavelength plate 581, a second wavelength plate 582, a third wavelength plate 583, and a fourth wavelength plate 584. The frame 580 holds a first wave plate 581, a second wave plate 582, a third wave plate 583, and a fourth wave plate 584. The first wave plate 581, the second wave plate 582, the third wave plate 583, and the fourth wave plate 584 are arranged in two rows and two columns with intervals between them.

The first wave plate 581, the second wave plate 582, the third wave plate 583, and the fourth wave plate 584 have different amounts of phase delay. Specifically, in the first wave plate 581, the amount of phase delay of the second polarized light wave with respect to the first polarized light wave is 0 [rad]. In the second wave plate 582, the amount of phase delay of the second polarized light wave with respect to the first polarized light wave is π/2 [rad]. In the third wave plate 583, the amount of phase delay of the second polarized light wave with respect to the first polarized light wave is π [rad]. In the fourth wavelength plate 584, the amount of phase delay of the second polarized light wave with respect to the first polarized light wave is 3π/2 [rad].

The four branched waves, which are the second polarized waves with different phase delays, enter the image sensor 60 via the second lens 59, the third lens 71, and the second polarizing plate 72. The image sensor 60 acquires an interference image by receiving the four branched waves on its imaging surface, and transmits data of the acquired interference image to the calculation unit 80 . The image sensor 60 is, for example, an image sensor such as a CCD image sensor or a CMOS image sensor.

The second polarizing plate 72 is an optical element that allows only light traveling in a specific direction (referred to as a "third direction") to pass through (transmit) for each branched wave in which a mutually different phase delay is applied to the second polarized light wave. . The polarization axis (transmission axis) in the second polarizing plate 72 and the first direction, which is the amplitude direction of the first polarized light wave, and the second direction, which is the amplitude direction of the second polarized light wave, are such that they intersect at an angle of 45°. It is located in Thereby, the ratio of the optical energy of the third direction component to the optical energy of the first polarized light wave before entering the second polarizing plate 72, and the ratio of the optical energy of the third direction component to the optical energy of the second polarized light wave before entering the second polarizing plate 72. The ratio of light energy is equivalent to

By passing through the second polarizing plate 72, the amplitude directions of the first polarized wave and the second polarized wave are aligned for each branch wave, and the first polarized wave and the second polarized wave interfere with each other. The light wave obtained by the interference between the first polarized wave and the second polarized wave is called an interference wave.

As a result, the image sensor 60 has an interference wave of the first branched wave, an interference wave of the second branched wave, an interference wave of the third branched wave, and an interference wave of the fourth branched wave in each of the four areas arranged in 2 rows and 2 columns. The interference wave of the branched wave is incident. Therefore, the image sensor 60 has an interference image with a phase delay amount of 0 as an interference wave of the first branch wave, an interference image with a phase delay amount of π/2 [rad] as an interference wave of the second branch wave, and an interference image of the third branch wave. An interference image with a phase delay amount π [rad] as a wave and an interference image with a phase delay amount 3π/2 [rad] as an interference wave of the fourth branched wave can be photographed.

The calculation unit 80 is electrically connected to the image sensor 60. The calculation unit 80 acquires a quantitative phase distribution of the sample 90 based on the four interference images captured by the image sensor 60. Specifically, the calculation unit 80 acquires data of the four interference images based on the signal received from the image sensor 60, and acquires a quantitative phase distribution of the sample 90 by calculation using gradient optical interference microscopy based on these four interference images. The specific calculation method used by the calculation unit 80 is described in, for example, the above-mentioned Non-Patent Document 1, and therefore details will be omitted.

The calculation unit 80 includes a storage unit that stores programs including algorithms for performing calculations based on gradient light interference microscopy, and a processor (for example, a CPU) that performs calculations according to the program. The storage unit includes, for example, a hard disk drive (HDD), read-only memory (ROM), or random access memory (RAM). Further, the calculation unit 80 may display the calculation results on a display (not shown) that is provided so as to be able to communicate with the calculation unit 80.

Here, the basic principle of this microscope 1 will be explained with reference to FIG. 5. FIG. 5 is a schematic diagram showing how reflected waves reflected from the sample surface are observed. In the example of FIG. 5, the incident light is separated by the differential interference prism 91 into a P-polarized light wave and an S-polarized light wave. Thereafter, the P polarized light wave and the S polarized light wave are incident on the sample 90 by the condenser lens 92 with a shear amount ΔS shifted in a direction perpendicular to the optical axis. As shown in FIG. 5, the S-polarized light wave corresponding to the P-polarized light incident on a certain position A1 on the sample 90 is made incident on a position A2 shifted by a shear amount ΔS in a predetermined direction from the position A1.

As a result, if the surface height of the sample 90 is not constant, the reflection height at position A1 is different from the reflection height at position A2. As a result, a phase difference occurs between the P-polarized reflected wave and the S-polarized reflected wave due to the difference in reflection height. Thereafter, the shear amount ΔS of the P-polarized reflected wave and the S-polarized reflected wave are aligned again, and the polarization directions are aligned to cause interference. At this time, the interference intensity is maximum when the phase difference is 0, 2π, and an integral multiple of 2π, and is minimum when the phase difference is π, 3π, and an integral multiple of 2π+π.

In this microscope 1, when the separation/combination is transmitted through the sample in the sample placement section 40, the first prism 32 and the condenser lens 33 cause the first polarized light wave and the second polarized light wave to be sheared in the direction perpendicular to the optical axis. The light is incident on the sample at a position shifted by ΔS and is transmitted through the sample. At this time, a phase difference occurs between the first polarized light wave and the second polarized light wave due to the difference in the state of the sample at each position. Here, the amount of phase delay of the second polarized light wave with respect to the first polarized light wave is assumed to be θ.

Thereafter, the objective lens 51 and the second prism 52 produce a composite wave in which the positional deviation between the first polarized light wave and the second polarized light wave is eliminated. At this time, the phase difference between the first polarized light wave and the second polarized light wave remains. Subsequently, the second polarized waves of the branched waves (first branched wave, second branched wave, third branched wave, and fourth branched wave) in which the composite wave is branched into four by the phase grating 56 are each split into two polarized waves. , 0, π/2, π, 3π/2 phase delays are given. As a result, the amount of phase delay of the second polarized light wave with respect to the first polarized light wave in each branched wave becomes θ, θ+π/2, θ+π, and θ+3π/2.

Finally, for each of the branched waves, the polarization directions of the first polarized light wave and the second polarized light wave are aligned by the second polarizing plate 72, so that the first polarized light wave and the second polarized light wave included in the composite wave are and interfere.

At this time, only qualitative evaluation is possible using only θ, in which the phase difference between the first polarized light wave and the second polarized light wave depends on the sample. On the other hand, quantitative evaluation can be performed by further adding a plurality of known phase differences 0, π/2, π, and 3π/2.

In the gradient light interference microscopy method described in Non-Patent Document 1, in order to perform imaging for four different amounts of phase delay, imaging must be performed four times. That is, since the imaging time is four times longer than that of the conventional differential interference microscope, it cannot be applied to dynamic samples.

On the other hand, in this microscope 1, the composite wave after irradiating the sample is branched into four light waves, and a mutually different amount of phase delay is added to each light wave. That is, in one imaging operation, imaging can be performed for four different amounts of phase delay. As a result, compared to conventional differential interference microscopes that can only perform qualitative evaluations, quantitative evaluations can be performed without increasing the imaging time.

<2. Second embodiment>
Below, a microscope 1A, which is a gradient light interference microscope according to a second embodiment of the present invention, will be described with reference to FIG. FIG. 6 is a schematic diagram showing the configuration of the microscope 1A. Similar to the microscope 1 of the first embodiment, this microscope 1A uses gradient light interference microscopy to image colorless and transparent objects such as cells, and uses one of two polarized waves with a shifted irradiation position. A plurality of phase delay amounts are added to the waveform, and the phase difference between the polarized lights is obtained from the intensity distribution of the combination of the two polarized waves.

As shown in FIG. 6, the microscope 1A includes a light source 20, an incident path 30A, a sample placement section 40, an exit path 50, an image sensor 60, and a calculation section 80. The microscope 1A of this embodiment irradiates light onto a sample placed in a sample placement section 40, and observes the reflected light using an imaging device 60. For this reason, the input path 30A from the light source 20 to the sample placement section 40 and the output path 50 from the sample placement section 40 to the image sensor 60 are arranged on straight lines that are perpendicular to each other.

The microscope 1A of this embodiment is different from the microscope 1 of the first embodiment in that the entrance path 30A has a half mirror 34, and accordingly, the entrance path 30A and the exit path 50 are arranged on different straight lines. The difference is that The other configurations are equivalent to the microscope 1 of the first embodiment. Regarding the reference numerals in FIG. 6, the same elements as the reference numerals in FIG. 1 are equivalent.

In the microscope 1A of this embodiment, the half mirror 34 is arranged between the condenser lens 33 and the sample arrangement section 40. The half mirror 34 transmits the light traveling from the condenser lens 33 toward the sample placement section 40, and reflects the light traveling from the sample placement section 40 toward the condenser lens 33 in the vertical direction.

As a result, in the microscope 1A, the light emitted from the light source 20 and passing through the first polarizing plate 31, the first prism 32, and the condenser lens 33 passes through the half mirror 34 and is incident on the sample placed in the sample placement section 40. The light reflected by the sample is then reflected vertically by the half mirror 34.

Then, the reflected light enters the objective lens 51 on the output path 50. The configuration from the emission path 50 to the image sensor 60 is the same as the microscope 1 according to the first embodiment.

In this way, with the microscope 1A of this embodiment, the reflected light of the sample can be observed using gradient light interference microscopy.

<3. Modified example>
Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments.

In the above embodiment, the branching mechanism that branches the combined light emitted from the second prism is a phase grating, but the present invention is not limited to this. The branching mechanism may be a diffraction grating other than a phase grating, such as an amplitude grating. Moreover, the branching mechanism may branch the light waves using other optical elements such as a half mirror.

Furthermore, in the above embodiment, the phase delay mechanism that delays the phase of one polarized light wave for each of the plurality of branched lights is a plurality of wavelength plates, but the present invention is not limited to this. For example, for each of the plurality of branched lights, a spatial optical phase modulator may be used to delay the phase of one polarized light wave.

Furthermore, the elements appearing in the above embodiments and modifications may be combined as appropriate to the extent that no contradiction occurs.

1, 1A Microscope 20

Light source

30, 30A Input path 31 First polarizing plate 32 First prism 33 Condenser lens 40 Sample placement section 50 Output path 51 Objective lens 52 Second prism 53 Color filter 54 First imaging lens 55 Second condenser Image lens 56 Phase grating 58 Phase delay section (wave plate)
60 Image sensor 72 Second polarizing plate 581 First wavelength plate 582 Second wavelength plate 583 Third wavelength plate 584 Fourth wavelength plate

Claims

A gradient light interference microscope that performs phase imaging using polarized waves,
an incidence path that allows light to enter the sample placement position from the light source;
an output path through which transmitted light or reflected light emitted from the sample placement position is incident on an image sensor;
including;
The incident path is, in order,
a first polarizing double-image prism that separates incident light into orthogonal first and second polarized waves;
a condenser lens disposed between the first polarizing double-image prism and the sample placement position;
has
The output path is, in order:
an objective lens into which the transmitted light or the reflected light emitted from the sample placement position is incident;
a second polarizing double-image prism that combines the first polarized light wave and the second polarized light wave;
a branching mechanism that branches the combined light emitted from the second polarizing double-image prism into a plurality of paths of three or more;
a phase delay mechanism that causes mutually different phase delays in the second polarized waves of each of the plurality of branched lights;
a polarizing plate that aligns the polarization directions of the first polarized light wave and the second polarized light wave of each of the plurality of branched lights;
Gradient light interference microscope.
The gradient light interference microscope according to claim 1,
A gradient light interference microscope, wherein the branching mechanism is a diffraction grating.
The gradient light interference microscope according to claim 2,
A gradient light interference microscope, wherein the branching mechanism is a phase grating.
4. The gradient light interference microscope according to claim 3,
A gradient light interference microscope, wherein the phase grating is arranged so that a Fourier spectrum distribution appears two-dimensionally in two rows and two columns on the imaging surface of the imaging element.
The gradient light interference microscope according to claim 4,
The phase delay mechanism is four wave plates arranged two-dimensionally in two rows and two columns,
A gradient light interference microscope in which the four wavelength plates have mutually different amounts of phase retardation.
The gradient light interference microscope according to any one of claims 1 to 5,
A gradient light interference microscope, wherein the phase delay mechanism is a plurality of wavelength plates corresponding to each of the branched lights.
The gradient light interference microscope according to any one of claims 1 to 6,
The branching mechanism branches the combined light into four or more paths,
The phase delay mechanism is a gradient light interference microscope, wherein the phase delay mechanism causes phase delays of 0, π/2, π, and 3π/2 in the second polarized light waves of each of the four branched lights.