JP2022139169A - microscope - Google Patents

Abstract

To provide a microscope capable of simultaneously acquiring quantitative phase images and bright-field microscope images.SOLUTION: The microscope is a combination of a transmission-type quantitative phase microscope that acquires quantitative phase images and a reflection-type bright-field microscope that acquires bright-field microscope images. The quantitative phase microscope is of off-axis type. The polarizing beam splitter PBS1 splits the light from a light source LD into s-polarized light and p-polarized light, one of which is on the measurement light path side and the other is on the reference light path side. The measurement light is condensed by a lens L2 and made incident on an objective lens OL1. A part of the measurement light with which an observation sample S is irradiated from the objective lens OL1 is reflected by the observation sample S and returns to the objective lens OL1, and is extracted by beam splitter BS2. The distance from the lens L2 to the back focal plane of the objective lens OL1 is set to match the focal length of the lens L2.SELECTED DRAWING: Figure 1

Description

The present invention relates to quantitative phase microscopy.

Quantitative phase microscopes are known that measure the phase delay when light passes through an observation sample by interference and image it (Non-Patent Document 1). As the structure of a quantitative phase microscope, an off-axis type (see Figure 9.1 of Non-Patent Document 1) and a common-path type (see Patent Document 1 and Figure 11.11 of Non-Patent Document 1) are known.

In the off-axis type, the light from the light source is split into reference light and measurement light, the measurement light is transmitted through the observation sample, and the transmitted measurement light and reference light are overlapped so as to form an angle to form interference fringes. It is generated to obtain phase information of the observed sample.

In the common-path type, part of the light transmitted through the observation sample is separated and passed through a pinhole, reference light is generated and overlapped again to generate interference fringes to obtain phase information of the observation sample.

JP 2008-292939 A

Quantitative Phase Imaging of Cells and Tissues, Gabriel Popescu, McGraw-Hill

However, in conventional off-axis quantitative phase microscopes, it has become impossible to simultaneously obtain quantitative phase images and normal microscope images (bright-field images and polarizing microscope images). Common-path quantitative phase microscopy can acquire normal images at the same time, but in common-path type, reference light is generated by passing part of the transmitted light of the observation sample through a multi-hole pinhole placed in the back focal plane. Therefore, it is necessary to either irradiate the observation sample with strong light, use an imaging device with high sensitivity, or take a long exposure time for the imaging device.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to enable simultaneous acquisition of a quantitative phase image and a normal microscope image in an off-axis quantitative phase microscope.

The present invention comprises a light source that emits parallel light, a light splitting section that splits the light from the light source into measurement light and reference light, and a measurement light emitted from the light splitting section to irradiate a sample to be observed with the measurement light. a first objective lens that passes the reflected measurement light again; a second objective lens that is arranged to face the first objective lens with the observation sample sandwiched therebetween; a second objective lens that passes the measurement light that has passed through the observation sample; a lens arranged between one objective lens for condensing the measurement light; a reflected light extraction section arranged between the lens and the first objective lens for extracting the measurement light reflected by the observation sample; and a reflected light extraction section a first imaging lens that forms an image of the measurement light extracted by the first imaging lens; a first photodetector that detects the measurement light imaged by the first imaging lens; and an image of the measurement light from the second objective lens. and a second imaging lens that combines the measurement light imaged by the second imaging lens with the reference light so that the optical axis of the measurement light and the optical axis of the reference light form an angle. and a second photodetector that detects the light combined by the optical combiner and detects interference fringes of the measurement light and the reference light, from the back focal plane of the first objective lens to the lens. is set to match the focal length of the lens.

In the present invention, the reflected light extraction unit may be a beam splitter, and the first light detection unit may acquire a bright field microscope image.

In the present invention, the reflected light extraction unit may be a polarizing beam splitter, and the first light detection unit may acquire a polarizing microscope image.

In the present invention, the distance from the back focal plane of the first objective lens to the lens may be 76.2 mm or less.

According to the microscope of the present invention, a quantitative phase image and a normal microscope image can be obtained simultaneously.

1 is a diagram showing the configuration of a microscope according to Example 1. FIG. 4 is a diagram showing a quantitative phase image measured by the microscope of Example 1. FIG. 4A and 4B are diagrams showing a quantitative phase image and a polarizing microscope image measured by the microscope of Example 1. FIG. The figure which showed the structure of the microscope of a modification.

Hereinafter, examples of the present invention will be described with reference to the drawings, but the present invention is not limited to the examples.

FIG. 1 is a diagram showing the configuration of the microscope of Example 1. FIG. In the microscope of Example 1, a transmission type quantitative phase microscope that acquires a quantitative phase image and a reflection type bright field microscope that acquires a bright field microscope image are combined, and a quantitative phase image and a bright field microscope image are simultaneously obtained. It is an affordable microscope. Also, the quantitative phase microscope is an off-axis type. A quantitative phase microscope is an upright type, and a bright field microscope is an inverted type.

As shown in FIG. 1, the microscope of Example 1 includes light sources LD and L, lenses L1 to L5, polarizing beam splitters PBS1 and PBS2, beam splitter BS1, objective lenses OL1 and OL2, imaging lens IL1, It has an IL2, an optical multiplexing element W, and imaging elements C1 and C2. The objective lens OL1 and the objective lens OL2 are arranged to face each other, and the observation sample S is arranged between the objective lens OL1 and the objective lens OL2.

The light source LD is a light source that emits laser light. The use of high-coherence laser light produces clearer interference fringes, facilitating the acquisition of quantitative phase images. The wavelength of the laser light emitted from the light source LD is arbitrary, and is 635 nm, for example. The laser light may be a continuous wave or a pulse wave, but in the case of a pulse wave, it is necessary to more strictly control the optical path difference between the measurement light and the reference light.

Instead of the light source LD, an LED, a halogen lamp, or the like that emits light with low coherence may be used, or white light may be used. By using light with low coherence, flickering due to interference can be suppressed, and a more accurate quantitative phase image can be obtained. However, it is necessary to sufficiently finely control the optical path difference between the measurement light and the reference light.

Light from the light source LD is collimated by the lens L1 and then enters the polarization beam splitter PBS1.

The polarizing beam splitter PBS1 splits the light from the light source LD into s-polarized light and p-polarized light, one of which is on the measurement light path side and the other on the reference light path side. Hereinafter, the light on the measurement light path side will be called measurement light, and the light on the reference light path side will be called reference light.

First, each configuration on the measurement optical path side and its operation will be described.

The beam splitter BS1 is a device that multiplexes and outputs two light beams that are input. The measurement light, which is one of the lights split into two by the polarization beam splitter PBS1, and the light from the light source L are input to the beam splitter BS1. The light source L is a halogen lamp. The light from the halogen lamp is condensed by the lens L5, passed through the aperture stop AS and the field stop FS, and after being reflected by the off-axis parabolic mirror OAPM with its angular distribution converted, enters the beam splitter BS1. is entered. In this way, by combining laser light with wide wavelength band and low coherence light from the halogen lamp, it is possible to acquire a normal microscope image with reduced speckle noise.

The measurement light output from beam splitter BS1 is condensed by lens L2, reflected by mirror M1, and then input to beam splitter BS2.

The beam splitter BS2 is arranged between the lens L2 and the objective lens OL1, and is a device for extracting the measurement light reflected by the observation sample S. The measurement light input from the mirror M1 is transmitted as it is to the objective lens OL1. , and the measurement light input from the objective lens OL1 is reflected and output to the image sensor C1.

The objective lenses OL1 and OL2 are arranged to face each other as shown in FIG. An observation sample S is arranged between the objective lens OL1 and the objective lens OL2. The objective lens OL1 is combined with the imaging lens IL1 to magnify the bright field microscope image to a predetermined magnification, and the objective lens OL2 is combined with the imaging lens IL2 to magnify the quantitative phase image at a predetermined magnification. It is intended to expand to A portion of the measurement light irradiated onto the observation sample S from the objective lens OL1 is reflected by the observation sample S and returns to the objective lens OL1, and a portion passes through the observation sample S and enters the objective lens OL2.

The measurement light reflected by the observation sample S is again input to the beam splitter BS2 through the objective lens OL1 and reflected. After that, after passing through a filter SF according to the purpose of measurement, it is further imaged by the imaging lens IL1 and input to the imaging device C1.

The imaging device C1 is a device that takes an image of light from the imaging lens IL1, and is, for example, a CCD camera. Thereby, a bright field microscope image can be acquired.

On the other hand, the measurement light transmitted through the observation sample S and input to the objective lens OL2 is input to the imaging lens IL2 through the objective lens OL2, formed into an image, and input to the optical multiplexing element W. FIG.

Next, each configuration on the reference optical path side and its operation will be described.

As shown in FIG. 1, the reference light, which is one of the lights output from the polarization beam splitter PBS1, is input to the polarization beam splitter PBS2 after being reflected by the mirror M2.

The optical path length of the reference light from the mirror M2 is controlled by the polarizing beam splitter PBS2, the quarter wave plate QWP, and the mirror M. The reference light input to the polarizing beam splitter PBS2 is directly transmitted through the polarizing beam splitter PBS2 and output, transmitted through the quarter-wave plate QWP, and then reflected by the mirror M2. The reference light reflected by the mirror M2 is again transmitted through the quarter-wave plate QWP and input to the polarization beam splitter PBS2. Since it passes through the quarter-wave plate QWP twice, the reference light input from the mirror M2 to the polarization beam splitter PBS2 does not pass through, but is reflected and output to the mirror M3. Also, this makes the polarization direction of the reference light coincide with the polarization direction of the measurement light. This makes it easier to generate interference fringes, and enables acquisition of a quantitative phase image with higher definition.

Here, the position of the mirror M2 is movable in its optical axis direction, and the optical path length of the reference light can be controlled by the position of the mirror M2. The optical path length of the reference light is controlled so that interference fringes of the measurement light and the reference light are generated.

The reference light reflected by the mirror M3 is condensed by the lens L3, passed through the pinhole PH, and returned to parallel light by the lens L4. By passing the reference light through the pinhole PH, the spatial intensity distribution of the reference light is made uniform and noise is reduced. Further, by passing through the pinhole PH, parallelization by the lens L4 can be performed with a higher degree of parallelism.

Light from the lens L4 is reflected by the mirror M4 after passing through the polarizing plate P, and is input to the optical multiplexing element W. FIG. By passing through the polarizing plate P, the light intensity of the reference light is matched with the light intensity of the measurement light, making it easy to generate interference fringes.

The optical multiplexing element W multiplexes the reference light from the mirror M4 and the measurement light from the imaging lens IL2 and outputs it. Further, the optical multiplexing element W multiplexes the optical axis of the reference light and the optical axis of the measurement light so as to form an angle. The multiplexed light is input to the imaging element C2. The optical multiplexing element W is, for example, a wedge plate beam sampler.

In Example 1, by using a wedge plate beam sampler, both the optical axis of the reference light and the optical axis of the measurement light are angled and the light is combined. may be attached. For example, by changing the condensing position when the measurement light from the light source LD is condensed on the rear focal plane of the objective lens OL1 by the lens L2, the angle formed by the optical axis of the reference light and the optical axis of the measurement light can be adjusted to may be controlled.

The imaging device C2 is a device that takes an image of the light from the optical multiplexing device W, and is, for example, a CCD camera. The reference light and the measurement light output from the optical multiplexing element W interfere with each other on the light receiving surface of the imaging element C2 to generate interference fringes. The imaging element C2 acquires this interference fringe image. The interference fringe image acquired by the imaging device C2 is input to the computer PC. A quantitative phase image is obtained by processing the interference fringe image with a computer PC.

For example, the processing in the computer PC is as follows. First, the interference fringe image is Fourier-transformed to transform the real-space image into a wavenumber-space image. A window function is applied to the image of the wavenumber space to impose a predetermined limit on the wavenumber space. Then, an inverse Fourier transform is performed to restore the image in the wavenumber space to the image in the real space. Logarithmic processing is then performed to evaluate the imaginary part to obtain phase information and a quantitative phase image.

Here, in order to generate interference fringes suitable for acquisition of a quantitative phase image, it is ideal that the wavefront curvatures of the measurement light and the reference light match, and that the polarization directions and light intensities are the same.

By matching the wavefront curvatures of the measurement light and the reference light, the interference fringes in the absence of the observation sample S can be linear, and when the interference fringe image is Fourier-transformed, it becomes point-like in wave number space. , a quantitative phase image can be acquired with higher definition.

Further, by equalizing the polarization directions and light intensities of the measurement light and the reference light, the brightness and darkness of the interference fringes can be made clearer, and a quantitative phase image can be obtained with higher definition.

As for the light intensity, the reference light is passed through the pinhole PH and the polarizing plate P to reduce the light intensity so that the reference light and the measurement light have the same light intensity. As for the polarization direction, the reference light and the measurement light are split using the polarization beam splitter PBS1, and the reference light is passed through the quarter-wave plate QWP twice, so that the polarization directions of the reference light and the measurement light match. I'm trying Therefore, matching the wavefront curvatures of the measurement light and the reference light becomes a problem.

A good method for matching the wavefront curvatures of the measurement light and the reference light is to make both the measurement light and the reference light input to the image sensor C2 parallel light. In other words, the radius of curvature of the wavefront is made to be infinite. In order to achieve this, a microscope combining an off-axis transmission type quantitative phase microscope and a reflection type bright field microscope as in Example 1 is configured as follows.

As already described, the reference light is passed through the pinhole PH with a sufficiently small opening, and then collimated by the lens L4 to obtain parallel light with a high degree of parallelism.

The measurement light is set so that the distance from the lens L2 to the rear focal plane of the objective lens OL1 matches the focal length of the lens L2. With this setting, the observation sample S is irradiated with parallel light, and the measurement light after passing through the imaging lens IL2 becomes parallel light.

By configuring as described above, both the reference light and the measurement light that are input to the optical multiplexing element W become parallel lights, so that the wavefront curvatures of the measurement light and the reference light can be matched.

Note that the fact that the distance from the lens L2 to the rear focal plane of the objective lens OL1 matches the focal length of the lens L2 does not mean that it matches completely, but to the extent that the effects of the present invention can be obtained. should match For example, the ratio of the focal length of the lens L2 to the distance from the lens L2 to the back focal plane of the objective lens OL1 is allowed to be 0.8 to 1.2.

The distance from the lens L2 to the rear focal plane of the objective lens OL1 (the focal length of the lens L2) should be as short as possible. This is because the diameter of the luminous flux of the measurement light with which the observation sample S is irradiated can be increased, and a quantitative phase image can be acquired over a wider area. Considering the actual product standard, cost, etc., it is preferable that the focal length of the lens L2 is 76.2 mm or less.

As described above, with the microscope of Example 1, a quantitative phase image and a bright-field microscope image can be obtained simultaneously. Further, with the microscope of Example 1, quantitative phase observation is possible with one optical system, from wide-range observation at low magnification to fine observation at high magnification.

In Example 1, a bright-field microscope image is acquired simultaneously with the quantitative phase image, but by changing the configuration as follows, a polarizing microscope image can be acquired simultaneously instead of the bright-field microscope image. . If it is desired to obtain a polarizing microscope image, the beam splitter BS1 should be replaced with a polarizing beam splitter PBS3 (see FIG. 4). In this case, it is possible to acquire a polarizing microscope image in the crossed Nicols arrangement.

In addition, in addition to between the lens L2 and the objective lens OL1, a beam splitter BS2 is inserted between the objective lens OL2 and the optical multiplexing element W to split a part of the measurement light, thereby obtaining a transmissive bright field. It is also possible to construct a microscope. Thereby, in addition to the reflection normal microscope image, a transmission normal microscope image is obtained.

Next, the result of analyzing the observed sample with the microscope of Example 1 will be described.

(Experiment 1)
First, an observation sample S in which two water droplets are present in paraffin oil is used, and an interference image obtained by the microscope of Example 1 and analyzed is shown.

FIG. 2(a) is an interference image acquired by the imaging device C2. As shown in the enlarged view, it can be seen that interference fringes at regular intervals corresponding to the carrier frequency are superimposed on the observed image.

FIG. 2(b) is a spatial frequency image obtained by Fourier transforming the interference image. A 0th-order spatial frequency image, which is a DC component, is obtained in the center, and 1st-order and −1st-order spatial frequency images are obtained at the upper left and lower right, respectively.

FIG. 2(c) is a spatial frequency image obtained by applying the shift rule of Fourier transform and filtering by a window function to the image of FIG. 2(b), extracting only the primary image and bringing it to the center.

The spatial frequency image of FIG. 2(c) is subjected to inverse Fourier transform to obtain the phase image of FIG. 2(d). However, in this phase image, since the phase is wrapped from -p to p, the phase unwrapping process is performed to smoothly connect the phases to obtain the phase image shown in FIG. 2(e). A phase difference of 2nπ (n is a natural number) can be distinguished if it is assumed that the phase changes continuously, and if the phase changes abruptly, it can be distinguished using white interference.

Finally, gradient compensation of the background phase is performed to obtain the phase image shown in FIG. 2(f). As shown in FIG. 2(f), it can be seen that two spherical water droplets are close to each other, and that there is a phase difference of about 8π between the paraffin oil and the water droplets. If the thickness of the observation sample S is known, it is possible to know a very small refractive index difference between paraffin oil and water droplets under predetermined measurement conditions (for example, a specific wavelength).

(Experiment 2)
A polarizing beam splitter PBS3 was used in place of the beam splitter BS1 so that a polarizing microscope image could be obtained instead of a bright field microscope image. The obtained polarizing microscope image is an image of the crossed Nicol arrangement. Then, a liquid crystal was used as the observation sample S, and a quantitative phase image and a polarizing microscope image were obtained at the same time. Liquid crystal materials are 4-Cyano-4'-pentylbiphenyl, 3,4-Difluoro-4'-(trans-4-pentylcyclohexyl)biphenyl, and 3,4-Difluoro-4'-(trans-4-propylcyclohexyl)biphenyl is a mixture of

FIG. 3(a) is an acquired quantitative phase image, and FIG. 3(b) is an acquired polarizing microscope image. As shown in FIG. 3, according to the microscope of Example 1, it turned out that a quantitative phase image and a polarizing microscope image can be acquired simultaneously.

The microscope of the present invention can be used for observing various samples.

LD, L: light sources L1 to L5: lenses PBS1 to PBS3: polarizing beam splitters BS1, BS2: beam splitters OL1, OL2: objective lenses IL1, IL2: imaging lenses W: optical multiplexing elements C1, C2: imaging elements

Claims (4)

a light source emitting parallel light;
a light splitting unit that splits light from the light source into measurement light and reference light;
a first objective lens that irradiates the observation sample with the measurement light through the measurement light from the light splitting unit and passes the measurement light reflected by the observation sample again;
a second objective lens arranged to face the first objective lens with the observation sample interposed therebetween, and through which the measurement light transmitted through the observation sample passes;
a lens disposed between the light splitting unit and the first objective lens for condensing the measurement light;
a reflected light extracting unit disposed between the lens and the first objective lens for extracting measurement light reflected by the observation sample;
a first imaging lens that forms an image of the measurement light extracted by the reflected light extraction unit;
a first photodetector that detects the measurement light imaged by the first imaging lens;
a second imaging lens that forms an image of the measurement light from the second objective lens;
an optical combiner for combining the measurement light imaged by the second imaging lens and the reference light so that the optical axis of the measurement light and the optical axis of the reference light form an angle;
a second photodetector that detects the light combined by the optical combiner and detects interference fringes of the measurement light and the reference light;
has
The distance from the back focal plane of the first objective lens to the lens is set to match the focal length of the lens,
A microscope characterized by: The reflected light extraction unit is a beam splitter,
The first light detection unit acquires a bright field microscope image,
2. The microscope according to claim 1, characterized in that: The reflected light extraction unit is a polarizing beam splitter,
The first photodetector acquires a polarizing microscope image,
2. The microscope according to claim 1, characterized in that: A microscope according to any one of claims 1 to 3, characterized in that the distance from the back focal plane of the first objective lens to the lens is 76.2 mm or less.

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