Phase-contrast microscope

Abstract

This invention provides a phase-contrast microscope that suppresses the loss of light intensity from higher-order diffraction and scattered light, thereby improving the clarity of fine images, and that can automatically remove noise light mixed into the laser beam. [Solution] The phase-contrast microscope apparatus 100 comprises a laser beam magnification parallel light configuration optical system 1 that forms a coherent magnification parallel laser beam including a laser light source 10, and a phase-contrast image creation optical system 2 which consists of a Fourier transform lens 14, an object introduction means 13, a spatial light modulator 15 positioned at the back focal plane of the Fourier transform lens 14, an inverse Fourier transform lens 16, a neutral density filter 17, and an electronic camera 18, with the F-number (focal length / effective aperture) of each lens 14 and 16 being F1.8 or less.

Description

Technical Field

[0005] , ,

[0001] The present invention relates to a phase contrast microscope device for simultaneously observing multiple items such as remote field large field of view, internal (deep) part observation, and real-time observation of the movement of the nucleus of living cells, etc.

Background Art

[0002] Conventionally, super-resolution microscopes such as electron microscopes, laser fluorescence microscopes, and probe microscopes using atomic force have been used for observing fine structures. However, it is impossible to observe in real time the behavior of viruses with a size of about 10 nm, the phagocytosis of cancer cells, the behavior of extracellular vesicles (EVs) with a size of about 10 nm, and the particle size distribution and behavior of ultra-fine bubbles in liquid using conventional super-resolution microscopes such as electron microscopes, laser fluorescence microscopes, and probe microscopes using atomic force.

[0003] It has long been believed that, due to the resolution limits of Abbe and Rayleigh, visible light microscopes cannot see anything finer than about 100 nm. As solutions, electron microscopes that perform observations using short wavelengths and laser fluorescence microscopes that stain samples for observation have been developed. However, these microscope observations require the sample to be thinly sliced and stained, which damages the sample.

[0004] That is, conventionally, optical microscopes have been considered to have the following two problems. 1. In an optical microscope, nothing finer than about 100 nm can be seen. 2. There is no optical imaging in a microscope that enables real-time, 3D, internal (deep) part, and movement simultaneous real-time observation of the shape without damaging the sample with super-resolution.

[0005] The applicant of this application has addressed the two challenges of the super-resolution microscope described above by proposing solutions in Patent Document 1 and Non-Patent Document 1. Patent Document 1 and Non-Patent Document 1 are technologies that advance conventional phase-contrast microscopes. These phase-contrast microscopes are used for observing transparent and thin samples such as living cells, and because they allow observation of structures even in an unstained state, they do not damage the sample and are suitable for observing cell dynamics.

[0006] Patent Document 1 discloses a phase contrast image inspection apparatus and a phase contrast image inspection method that use a centrally perforated λ / 4 plate as an optical filter, using zero-order diffracted light as reference light and separating and observing higher-order diffracted and scattered light as object light. This invention makes it possible to simultaneously observe multiple items such as large field of view in remote areas, internal (deep) observation, and real-time observation of motion with super-resolution of several tens of nanometers, without damaging the sample by slicing or staining, which was not possible with conventional techniques.

[0007] Non-patent document 1 discloses an example in which the technology of patent document 1 is applied using a wide-aperture lens, enabling simultaneous observation of multiple parameters such as large-field, deep-field, and real-time motion observation with unstained super-resolution of several tens of nanometers. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 6603588 [Non-patent literature]

[0009] [Non-Patent Document 1] I. Shimizu, et al, “Contrast-tuneable microscopy for single-shot real-time imaging.”Eur. Phys. J. Appl. Phys.91, 30701(2020) [Overview of the project] [Problems that the invention aims to solve]

[0010] Patent Document 1 and Non-Patent Document 1 solve the two problems mentioned above, but Patent Document 1 and Non-Patent Document 1 have the following problems: (1) Because higher-order diffraction and scattered light becomes circularly polarized, when adjusting the intensity of object light and reference light with a polarizing plate placed in front of the camera, some of the object light is lost, resulting in a slight lack of image clarity. Also, (2) When noise light is mixed with the light source laser, it is difficult to block the effect of that noise light with the filter.

[0011] In view of two conventional problems and two new problems, the present invention aims to provide a phase-contrast microscope that enables the suppression of the loss of light intensity of object light, which is high-order diffracted and scattered light, thereby obtaining sharpness of fine images, and also has a function to automatically eliminate noise light and other impurities mixed into the light source laser light.

[0012] In other words, the objective is to provide a phase-contrast microscope apparatus capable of the following: 1. Observe the shape of samples smaller than approximately 100 nm using an optical microscope without damaging them. 2. Super-resolution allows for real-time observation of 3D, internal (deep) structures and motion. 3. It can reduce the loss of light intensity of object light, increasing the clarity of fine images. 4. Provide an automatic function to eliminate noise in optical images, etc. [Means for solving the problem]

[0013] To solve the above problems, the phase-contrast microscope apparatus of the present invention includes a laser beam magnification parallel light configuration optical system that forms a coherent magnification parallel laser beam including a laser light source, A Fourier transform lens placed in a coherent parallel laser beam and , on the side of the laser light source from the Fourier transform lens An object introduction means provided for introducing an object; a spatial light modulator provided on the back focal plane of the Fourier transform lens to adjust the intensity of zero-order light and higher-order diffracted and scattered light; an inverse Fourier transform lens provided with the back focal plane of the Fourier transform lens as the front focal plane to collect the zero-order light and higher-order diffracted and scattered light that has passed through the spatial light modulator; and the inverse Fourier transform lens Closer to the electronic camera side or the spatial light modulator side A light-reducing filter installed and an optical image formed on the light-gathering surface of the inverse Fourier transform lens are installed to capture the optical image. The aforementioned The system comprises an electronic camera and a phase-difference image creation optical system, each configured with the cameras positioned on the optical axis. Large-aperture lenses are used for the aforementioned Fourier transform lens and the aforementioned inverse Fourier transform lens. The system captures a phase-difference object interference image formed by zero-order light and higher-order diffracted / scattered light with adjusted intensity differences for an object located outward in a direction perpendicular to the optical axis, over a wide field of view, and the F-number (focal length / effective aperture) of the Fourier transform lens and the inverse Fourier transform lens are F1.8 or less. [Effects of the Invention]

[0014] The phase-contrast microscope apparatus of the present invention allows for the observation of the shape of samples smaller than 100 nm without damaging them using an optical microscope, and enables real-time observation of 3D, internal (deep), and motion using super-resolution. Furthermore, the phase-contrast microscope apparatus of the present invention can suppress the loss of light intensity of object light, which is high-order diffraction and scattered light, in order to obtain clarity of fine images, and can also automatically eliminate noise light mixed in with the laser light source. [Brief explanation of the drawing]

[0015] [Figure 1] (A) is an explanatory diagram showing the general configuration of a phase-contrast microscope system using a large-aperture lens, and (B) is an explanatory diagram showing the patterns of scattered and diffracted light. [Figure 2] Figure 1 is an explanatory diagram showing the light-gathering (A) and image-forming processes of the large-aperture lens in the phase-contrast microscope apparatus. [Figure 3] This diagram illustrates the difference in image resolution due to the aperture ratio of the lenses in a phase-contrast microscope. (A) shows an example where large-aperture lenses are used for both the Fourier transform lens and the inverse Fourier transform lens, and (B) shows an example where an aperture is placed on the large-aperture lens of the Fourier transform lens and a large-aperture lens is used for the inverse Fourier transform lens. [Figure 4](A) of the phase contrast microscope apparatus according to an embodiment of the present invention is an explanatory diagram showing the configuration of the optical system, and (B) is an explanatory diagram showing the polarization direction and intensity of the laser light. [Figure 5] (A) of the conventional phase contrast microscope apparatus is an explanatory diagram showing the configuration of the optical system, and (B) is an explanatory diagram showing the polarization direction of the laser light. [Figure 6] It is an explanatory diagram showing the configuration of the optical system of a phase contrast microscope apparatus equipped with a secondary optical system to increase the optical magnification and resolution, and an explanatory diagram showing the polarization direction of the laser light. [Figure 7] It is an image of monodisperse transparent standard particles observed with the phase contrast microscope apparatus according to an embodiment of the present invention. [Figure 8] It is an explanatory diagram showing a size calibration method for resolution inspection of the phase contrast microscope apparatus according to an embodiment of the present invention. [Figure 9] It is an image obtained by observing in real time the behavior of super-resolution microparticles and their interaction with cells using the phase contrast microscope apparatus according to an embodiment of the present invention. [Figure 10] It is an explanatory diagram showing a schematic configuration of a phase contrast microscope apparatus in which an evanescent field is set.

Embodiments for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention (hereinafter referred to as examples) will be described based on the drawings. In the following figures, the same reference numerals are given to common parts, and duplicate explanations for the parts with the same reference numerals are omitted.

[0017] The phase-contrast microscope apparatus 100 of the present invention comprises a laser beam expanding parallel light configuration optical system (1) that forms a coherent expanding parallel laser beam including a laser light source 10 as shown in Figures 1(A), 3(A), and 4(A); a Fourier transform lens 14 placed in the coherent parallel laser beam and an object introduction means 13 provided in front of it for introducing an object; a spatial light modulator (spatial light modulation filter) 15 provided on the back focal plane of the Fourier transform lens 14 for adjusting the intensity of zero-order light and higher-order diffracted and scattered light; an inverse Fourier transform lens 16 that is set with the back focal plane of the Fourier transform lens 14 as the front focal plane and collects the zero-order light and higher-order diffracted and scattered light that has passed through the spatial light modulator 15; a neutral density filter 17 placed behind or in front of the inverse Fourier transform lens 16; and an electronic camera 18 installed to capture an optical image formed on the focusing surface of the inverse Fourier transform lens 16, all of which are arranged on the optical axis to create a phase-contrast image. As shown in Figure 1(A), the neutral density filter 17 is preferably placed in front of the electronic camera 18 behind the inverse Fourier transform lens 16, but it may also be placed in front of the inverse Fourier transform lens 16 and behind the front focal point of the inverse Fourier transform lens.

[0018] With the phase-contrast microscope apparatus 100 configured in this way, it is possible to capture a phase-contrast object interference image with a wide field of view, formed by zero-order light and higher-order diffracted and scattered light with adjusted intensity differences, for objects located outward in a direction perpendicular to the optical axis. The laser beam magnified parallel light configuration optical system (1) consists of a laser light source 10 and lenses 11 and 12 installed after it. The coherent light emitted from the laser light source 10 is emitted as a parallel beam through lenses 11 and 12.

[0019] Large-aperture lenses are used for both the Fourier transform lens 14 and the inverse Fourier transform lens 16. Large-aperture lenses have a large diameter and can capture a large amount of light. The F-number is expressed as focal length / effective aperture, and lenses with a small F-number have a large aperture. By using large-aperture lenses, the loss of light intensity of object light, which is high-order diffracted and scattered light, can be suppressed, and fine image clarity can be obtained. The F-number is not particularly limited as long as a particularly clear image can be obtained, but in this embodiment, large-aperture lenses with an F-number of 1.8 or less, preferably 1.4 or less, are used. By focusing and imaging with such large-aperture lenses, even the shape of fine samples of about 100 nm or less can be clearly observed due to high-order diffracted and scattered light.

[0020] The spatial light modulator (SLM) 15 has the function of transmitting higher-order diffracted light and is connected to a terminal device (such as a personal computer) 20 for image processing and data processing. By rewriting and deleting the image displayed there, the intensity of zero-order light and higher-order diffracted and scattered light can be adjusted. In this embodiment, noise light and other particles mixed in with the light source laser light are automatically removed. By providing an automatic removal function for noise light images, observation can be performed efficiently. It is also possible to manually adjust the removal of noise light and other particles.

[0021] Figures 1 and 2 illustrate the basic principle by which the phase-contrast microscope apparatus 100 of the present invention can obtain super-resolution images. Figure 1(A) is an explanatory diagram showing the schematic configuration of the phase-contrast microscope apparatus 100, and (B) is an explanatory diagram showing the scattered and diffracted light patterns. Figure 2(A) is an explanatory diagram showing the focusing of a large-aperture lens, and (B) is an explanatory diagram showing the image formation.

[0022] As shown in Figures 1(A) and 1(B), when a laser beam of uniformly coherent, parallel intensity is shone onto an object (sample microparticles), the light that does not hit the object and some of the light that passes through the sample travels in a straight line, while the scattered and diffracted light, including refraction from the sample, spreads outward from the direction of the shone light. Among the scattered and diffracted light from the object, the transmitted straight-traveling light is zero-order light (0th-order diffracted light), and the light that is scattered and refracted and spreads outward is higher-order diffracted and scattered light (1st-order diffracted light, 2nd-order diffracted light). The higher-order diffracted and scattered light contains shape information about the shape and size of the sample object, and the smaller the sample object, the more the higher-order diffracted and scattered light spreads outward from the optical axis. In Figure 1(A), these zero-order light and higher-order diffracted and scattered light are focused by a large-aperture Fourier transform lens 14 (FT lens), and only the zero-order light is blocked or attenuated by a spatial light modulator 15 installed on the back focal plane of the FT lens 14. As mentioned above, the spatial light modulator 15 is connected to the terminal device 20, and the intensity of zero-order light and higher-order diffracted and scattered light can be adjusted on its screen.

[0023] By imaging with an inverse Fourier transform lens (iFT lens) 16, which is positioned with the back focal plane of the Fourier transform lens 14 as the front focal plane, as described above, even the shape of minute samples of about 100 nm or less can be clearly observed due to high-order diffraction and scattered light. The left figure of Figure 2(A) shows an example in which a large-aperture lens is used as the Fourier transform lens 14, and the right figure shows an example in which it is not used. The left figure of Figure 2(B) shows an example in which a large-aperture lens is used as the inverse Fourier transform lens 16, and the right figure shows an example in which it is not used. As shown in Figures 2(A) and (B), especially when the sample object is made of fine particles, high-order diffraction and scattered light spread outward, so a super-resolution image can be obtained by focusing and imaging with a large-aperture lens.

[0024] Figure 3 is an explanatory diagram showing the difference in image resolution depending on the aperture ratio of the lenses of the phase-contrast microscope apparatus 100. (A) shows an example in which large-aperture lenses are used for the Fourier transform lens 14 and the inverse Fourier transform lens 16, and (B) shows an example in which an aperture is placed on the large-aperture lens of the Fourier transform lens 14 and a large-aperture lens is used for the inverse Fourier transform lens 16. As shown in Figure 3, triangles and circles of different thicknesses are used as sample objects. As shown in Figure 3(A), when large-aperture lenses are used for the Fourier transform lens 14 and the inverse Fourier transform lens 16, the acquired image has high resolution, and the shapes of the triangle and circle can be observed with precision. On the other hand, as shown in Figure 3(B), when a large-aperture lens is not used for the Fourier transform lens 14, the acquired image has low resolution, and the shapes of the triangle and circle are blurred. From the comparison between Figure 3(A) and (B), it can be seen that lenses with a large aperture ratio are necessary for the Fourier transform lens 14 and the inverse Fourier transform lens 16 in order to acquire fine images of 100 nm or less.

[0025] Figure 4 shows an explanatory diagram of the optical system configuration for the phase-contrast microscope apparatus 100 (A) and an explanatory diagram showing the polarization direction and intensity of the laser light (B). Figure 5 shows an explanatory diagram of the optical system configuration for the conventional phase-contrast microscope apparatus 200 (A) and an explanatory diagram showing the polarization direction of the laser light (B). Figure 4(B) shows the scattered and diffracted light pattern from a sample introduced into the P1 plane and the image taken at P3, as well as the polarization directions of the irradiated light and scattered / diffracted light in the P1-P3 planes. In Figure 4(B), dashed arrows indicate irradiated or 0th-order diffracted light, and solid arrows indicate higher-order diffracted light. The same applies to Figure 5(B).

[0026] The phase-contrast microscope apparatus 100 of the present invention is equipped with a spatial light modulator 15 necessary for efficient separation of zero-order light and higher-order diffracted / scattered light, as well as automatic image processing of noise light, etc. However, conventional phase-contrast microscope apparatus 200 (Patent Document 1) does not have a spatial light modulator 15, but has a λ / 4 plate (filter) 19. The phase-contrast microscope apparatus 100 in Figure 4(A) is equipped with a spatial light modulator 15 connected to a terminal device (such as a personal computer) 20 for image processing and data processing, enabling efficient separation of higher-order diffracted / scattered light containing information about the shape and size of the sample object from the zero-order light that serves as the reference light, as well as light intensity adjustment and the ability to select and erase or enhance images of arbitrary shapes.

[0027] The difference in effect is shown in the difference in the polarization direction of the laser light in Figure 4(B) and Figure 5(B). In Figure 4(B), the higher-order diffracted and scattered light is linearly polarized, but in Figure 5(B), it is circularly polarized. When the higher-order diffracted and scattered light is circularly polarized, there is a disadvantage that some of the object light is lost when adjusting the intensity of the object light and the reference light with the polarizing plate 17 installed in front of the camera 18, resulting in a slight lack of image sharpness. The phase-contrast microscope apparatus 100 of the present invention solves this problem. Furthermore, in the conventional phase-contrast microscope apparatus 200 shown in Figure 5(A), there was a disadvantage that if noise light was mixed in with the light source laser 10, it was difficult to block the effect of that noise light with the λ / 4 plate (filter) 19, but the phase-contrast microscope apparatus 100 of the present invention solves this problem by being equipped with a spatial light modulator 15.

[0028] Figure 6 is an explanatory diagram showing the optical system configuration of a phase-contrast microscope with a secondary optical system to increase optical magnification and improve resolution, and an explanatory diagram showing the polarization direction of the laser light. The resolution of a microscope is determined by the resolution of the image sensor (pixel) of the microscope's imaging camera. Therefore, in order to increase the resolution of a microscope, it is necessary to increase the optical magnification of the microscope. To increase the optical magnification of the super-resolution microscope, as shown in Figure 6, this microscope uses a lens system with a Fourier transform lens 14 as the objective lens and an inverse Fourier transform lens 16 behind it as the primary optical system, and a secondary optical system with a Fourier transform lens 14, a spatial light modulation filter 15, and an inverse Fourier transform lens 16 behind it to form the optical system. With this configuration, a resolution of more than 15.7 nm / pixel was obtained, and transparent standard particles of 40 nm in liquid were observed.

[0029] Figure 7 shows images of monodisperse transparent standard particles observed with the phase-contrast microscope apparatus 100 of the present invention. Specifically, these are images of transparent standard-sized polystyrene latex particles with a diameter of 40 nm observed with the microscope 100 of this embodiment. The left figure shows an image from a hemocytometer, and the right figure shows an image of a group of 40 nm PL particles in liquid on a glass slide.

[0030] Figure 8 is an explanatory diagram showing the size calibration method for resolution inspection of the phase-contrast microscope apparatus 100. Calibration experiments of the phase-contrast microscope apparatus 100 were performed using standard particles of transparent polystyrene latex with guaranteed size. Figure 8 shows an example of the size calibration method for microscope images. A sample of polystyrene latex standard particles suspended in ultrapure water is placed on the sample surface of the microscope and an image is taken. A line profile is drawn on the line of the image of the fine particles arranged in the captured image (image of 40 nm PL particles suspended in ultrapure water), as shown in the upper left of Figure 8 (vertical axis is Gray Value, horizontal axis is pixels), and the particle diameter is calculated from the width of the line profile. Note that the Gray Value is a value that indicates the brightness of each pixel in the grayscale image.

[0031] As shown in the upper right of Figure 8, the broken line of light intensity along the diameter of the particle image represents the image intensity for each pixel, and the particle diameter is calculated from the width of the line profile, as shown in the middle right of the figure. The table at the bottom of Figure 8 shows the results of calibration experiments performed using standard PL particles of 100 nm, 80 nm, and 40 nm. From these results, it was clearly demonstrated that the phase-contrast microscope apparatus 100 of the present invention currently has a resolution of 40 nm. The resolution of the microscope at this time was 15.5 nm / pixel.

[0032] Figure 9 shows images of the behavior of super-resolution microparticles and their interaction with cells observed in real time using the phase-contrast microscope 100. Specifically, it shows how 80 nm diameter transparent polystyrene latex particles move around living cells. As described above, by using the phase-contrast microscope 100 of the present invention, it is possible to observe the shape of a fine sample of 40 nm without damaging it, and to observe 3D, internal (deep), and motion in real time using super-resolution.

[0033] Figure 10 is an explanatory diagram illustrating the schematic configuration of a phase-contrast microscope system with an evanescent field. An evanescent field is a localized electromagnetic field that arises under specific conditions, and is a special field in which the propagation of electromagnetic waves is restricted. When near-field light is shone on a minute object in an evanescent field, the directional intensity of scattered and reflected light is increased, making it possible to observe the structure and molecular motion near the surface with high sensitivity. As shown in Figure 10, by totally reflecting strong parallel laser light off the glass plate of the object introduction means (sample placement area) 13 to create an evanescent field in the object introduction means (sample placement area) 13, a measurement field of view composed of vertically irradiated parallel laser light is constructed. Due to the influence of the evanescent field, scattered light from a sample placed in the measurement field of view can be observed with magnified detail, resulting in a sample image of several nanometers in size. In this way, the resolution of the sample image can be improved by adding an evanescent field.

[0034] As described above, the phase-contrast microscope apparatus of the present invention allows for the observation of the shape of samples smaller than 100 nm without damaging them using an optical microscope, and enables real-time observation of 3D, internal (deep), and motion using super-resolution. Furthermore, the phase-contrast microscope apparatus of the present invention can suppress the loss of light intensity of object light, which is high-order diffraction and scattered light, in order to obtain clarity of fine images, and can also automatically eliminate noise light mixed in with the laser light source.

[0035] The phase-contrast microscope apparatus described above is merely an example, and its configuration and other aspects can be modified as appropriate without departing from the spirit of the invention. [Explanation of symbols]

[0036] 10 Laser light source 11 lenses 12 lenses 13 Object introduction means 14. Fourier transform lens 15. Spatial Light Modulator 16 Inverse Fourier Transform Lens 17 Neutral Density Filter 18 Electronic Cameras 19 λ / 4 plate (filter) 20 Terminal devices 100 Phase-contrast microscopes 200 Conventional Phase Contrast Microscopes

Claims

1. A laser beam expansion parallel optical system that forms a coherent expansion parallel laser beam including a laser light source, A phase-difference image creation optical system is configured with the following elements, each positioned on the optical axis: a Fourier transform lens placed in a coherent parallel laser beam; an object introduction means provided on the laser light source side of the Fourier transform lens for introducing an object; a spatial light modulator provided on the back focal plane of the Fourier transform lens for adjusting the intensity of zero-order light and higher-order diffracted and scattered light; an inverse Fourier transform lens positioned with the back focal plane of the Fourier transform lens as the front focal plane for focusing the zero-order light and higher-order diffracted and scattered light that has passed through the spatial light modulator; a neutral density filter provided on the electronic camera side or the spatial light modulator side of the inverse Fourier transform lens; and an electronic camera provided for capturing an optical image formed on the focusing surface of the inverse Fourier transform lens. Large-aperture lenses are used for the aforementioned Fourier transform lens and the aforementioned inverse Fourier transform lens. A phase-contrast microscope apparatus characterized by capturing a phase-contrast object interference image formed by zero-order light and higher-order diffracted and scattered light with adjusted intensity differences for an object located outward in a direction perpendicular to the optical axis, over a wide field of view, and having F-numbers (focal length / effective aperture) of the Fourier transform lens and the inverse Fourier transform lens, respectively, of F1.8 or less.

2. The phase-contrast microscope apparatus according to claim 1, characterized in that the object introduction means is provided with means for setting an evanescent field.

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