Super-focusing microsphere lens and super-resolution imaging system

Abstract

The invention discloses a super-focusing microsphere lens based on phase modulation and a super-resolution imaging system. The system comprises a monochromatic light source used for illumination, a lens used for collimating and converging light, an optical small hole, a beam splitter, a phase modulator, a microsphere lens, an imaging sample, a three-dimensional displacement table and a photomultiplier. Different from a common micro-sphere lens super-resolution imaging system, the micro-sphere lens super-resolution imaging system has the advantages that the phase modulator is additionally arranged above the micro-sphere lens, focusing light spots of the micro-sphere lens are smaller through implementation of a specific phase of an incident light field, and higher imaging resolution can be obtained when the micro-sphere lens super-resolution imaging system is applied to a confocal scanning imaging light path. Compared with other super-resolution imaging technologies, the method does not need fluorescent staining and image post-processing, and has very strong compatibility with a confocal optical microscope.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopy imaging, and particularly relates to a superfocusing microsphere lens based on phase modulation and a super-resolution microscopy imaging system implemented by using the microsphere lens. Background Art

[0002] Generally, the imaging resolution of an optical microscope is jointly determined by the numerical aperture of its objective lens and the wavelength of the illumination light source. Therefore, people can improve the imaging resolution of the system by shortening the wavelength of the illumination light and increasing the numerical aperture of the objective lens. However, a shorter wavelength means higher photon energy, which causes higher light damage to the imaged sample, especially for some biological samples; and the numerical aperture of the objective lens is jointly determined by the maximum light acceptance angle of the objective lens and the refractive index of the immersion medium. In the case where the light acceptance angle cannot be further increased, only the refractive index of the immersion medium can be increased. When the sample is placed in a liquid, the immersion medium changes from air to liquid, and its refractive index can be greatly increased. However, due to the limited types of existing materials, among the existing immersion liquids, there are few liquids with a refractive index exceeding 2.0. Therefore, it is very difficult to further increase the numerical aperture of the objective lens.

[0003] In the existing super-resolution microscopy imaging technologies, fluorescence microscopes represented by STED, STORM, and PALM have achieved nanoscale imaging resolution, which has greatly promoted the rapid development of biomedicine. However, the above technologies are helpless for non-biological samples that cannot be stained. In non-fluorescent super-resolution microscopy imaging technologies, the structured light illumination technology is the most mature. During its imaging process, it is necessary to use structured light with different directions and different phases to illuminate the sample, and then invert the surface topography of the sample according to the Moiré fringes received by the objective lens. Therefore, the imaging process is a process of inversion calculation based on multiple frames of images, and it is very difficult to achieve real-time imaging. The microsphere-assisted microscopy imaging technology is a new super-resolution imaging scheme. It can break through the optical diffraction limit, improve the imaging resolution of the microscope objective lens, and also improve the system magnification by using the near-field focusing and near-field imaging characteristics of the microsphere lens. It has a simple structure, convenient operation, and can be compatible with ordinary optical microscopes. Therefore, it has great application potential. However, the super-resolution imaging ability of the microsphere lens cannot meet the increasingly high requirements for ultra-precise imaging and detection. Although by optimizing parameters such as the refractive index, size, and shape of the microsphere, the focal spot size of the microsphere lens can be further compressed and its imaging resolution can be improved, at the same time as compressing the focal spot, the light intensity of the focal spot sidelobe will also be increased, thus affecting the imaging quality. Therefore, it is very difficult for the microsphere lens to be continuously optimized and improved by itself.

[0004] This patent combines the super-resolution imaging characteristics of microsphere lenses with optical phase modulation technology to propose a new type of optical super-resolution microscopy imaging technology. This solution will utilize the modulation effect of phase on the focal spot of the microsphere lens to further compress the focal spot size of the microsphere lens. Applying it to the confocal imaging optical path can further improve the imaging resolution of the system. Compared with the existing microsphere-assisted super-resolution imaging system (CN201110139222.8), the core technology of this patent is to add a phase modulator above the microsphere lens to control the phase distribution of the incident light, thereby greatly improving the focusing performance and super-resolution imaging performance of the microsphere lens. In addition, to overcome the destructive effect of high-intensity sidelobes on the imaging quality, the confocal imaging optical path is selected in this invention, which can greatly shield the influence of sidelobes in the imaging result. Summary of the Invention

[0005] To further improve the imaging resolution of the microsphere-assisted super-resolution imaging system, this invention proposes a super-focusing microsphere lens and a super-resolution imaging system based on phase modulation. The system is jointly composed of a monochromatic illumination light source, an optical pinhole, a lens, a beam splitter, a phase modulator, a microsphere lens, an imaging sample, a three-dimensional displacement stage, and a photomultiplier tube. Compared with the ordinary microsphere-assisted super-resolution imaging system, the innovation of this invention lies in the use of the phase modulator. Through the phase modulation function of the phase modulator, the light fields passing through two adjacent circular phase modulation units have a phase difference of π. After passing through the microsphere lens, multiple different focal spots will be formed, and then the interference cancellation between different focal spots is used to compress the lateral size of the central focal spot, and a finer focusing focal spot can be obtained. Applying it to the confocal imaging system can further break through the diffraction limit and improve the imaging resolution of the system. By precisely modulating the phase of the incident light field above the microsphere lens, this invention can achieve a finer focusing spot, and applying it to the confocal imaging optical path can greatly improve the imaging resolution of the system.

[0006] To achieve the above object, the technical solution adopted by this invention is as follows: A superfocusing microsphere lens and super-resolution imaging system based on phase modulation, which are jointly composed of a monochromatic illumination light source, an optical pinhole, a lens, a beam splitter, a phase modulator, a microsphere lens, an imaging sample, a three-dimensional displacement stage, and a photomultiplier tube. Similar to the ordinary confocal imaging optical path, in the present invention, optical pinholes are respectively arranged on the focal planes of two lenses to suppress the influence of the sidelobe intensity in the focusing focal spot of the microsphere lens on the imaging quality. The difference lies in that in the present invention, a microsphere lens is used to replace the original microscope objective lens, and the superfocusing ability of the microsphere lens can be utilized to improve the imaging resolution. In addition, to further break through the resolution limit of the microsphere lens itself, a phase modulator is added above the microsphere lens in the present invention. By regulating the phase of the incident light field of the microsphere lens, a finer focusing focal spot and a higher imaging resolution can be achieved. Specifically, each circular modulation unit in the phase modulator can transmit a beam of light, and each beam of light will form a focusing focal spot after passing through the microsphere. When the distances between adjacent two phase modulation units are very close, a part of the two generated focal spots will overlap in space to form a larger focal spot. However, if the phase difference of the light beams emitted by each phase modulation unit can be controlled to ensure that the light beams emitted by adjacent two modulation units have a phase difference of π, then the focal spots of the two will cancel each other out in the overlapping area, that is, the intensity of the central area is zero. As the two get closer and closer, the mutual extrusion phenomenon of the focusing spots becomes more obvious. Using the above principle, the focal spot size of the microsphere lens can be fully compressed, thereby achieving a higher imaging resolution.

[0007] Compared with the prior art, the present invention has the following beneficial effects: A superfocusing microsphere lens and super-resolution imaging system based on phase modulation, which are jointly composed of a monochromatic illumination light source, an optical pinhole, a lens, a beam splitter, a phase modulator, a microsphere lens, an imaging sample, a three-dimensional displacement stage, and a photomultiplier tube. Compared with an ordinary confocal scanning optical microscope, the present invention uses a microsphere lens to replace the traditional microscope objective lens, and higher imaging resolution can be obtained. Compared with the existing microsphere-assisted super-resolution imaging system, the present invention introduces a phase modulator. By modulating the phase of the incident light field of the microsphere lens, the focal spot size of the microsphere lens can be further compressed, thereby achieving a higher resolution. Compared with other super-resolution imaging technologies, this solution does not require multiple imaging, does not require image post-processing, and can be well compatible with a confocal optical microscope, and can be applied to some industrial measurement fields that require large area, high precision, and high speed. Description of the Drawings

[0008] Figure 1 It is a schematic diagram of an optical microscopic imaging system based on mask phase modulation proposed by the present invention. Wherein: 1. Monochromatic illumination light source; 2. Optical pinhole; 3. Lens; 4. Beam splitter; 5. Phase modulator; 6. Microsphere lens; 7. Imaging sample; 8. Three-dimensional displacement stage; 9. Photomultiplier tube.

[0009] Figure 2 (a) is a schematic diagram of the operation of the phase modulator and the microsphere lens in the present invention, where: 1. Monochromatic illumination light source; 2. Phase modulator; 3. Microsphere lens. After passing through the phase modulator, the light enters the microsphere lens with a specific phase and is then focused by the microsphere lens into an ultra-fine focal spot; Figure 2 (b) is a schematic structural diagram of the one-dimensional phase modulator in the present invention, which is composed of a transparent substrate 4 and three circular phase modulation units 5; Figure 2 (c) is a schematic structural diagram of the two-dimensional phase modulator in the present invention, which has a total of five circular phase modulation units.

[0010] Figure 3 is the original focusing effect diagram of the microsphere lens without using the phase modulator in the present invention, Figure 3 (a) is the light intensity distribution diagram on the focal plane; Figure 3 (b) is the light intensity distribution curve on the white dotted line.

[0011] Figure 4 is the focusing effect diagram of the microsphere lens after using the one-dimensional phase modulator in the present invention, Figure 4 (a) is the light intensity distribution diagram on the focal plane; Figure 4 (b) is the light intensity distribution curve on the white dotted line.

[0012] Figure 5 is the focusing effect diagram of the microsphere lens after using the two-dimensional phase modulator in the present invention, Figure 5 (a) is the light intensity distribution diagram on the focal plane; Figure 5 (b) is the light intensity distribution curve on the white dotted line. Detailed implementation manners

[0014] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the following further details the present invention in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not limit the present invention. The detailed description is as follows.

[0015] Figure 1Schematic diagram of the structure of the superfocusing microsphere lens and super-resolution imaging system based on phase modulation proposed by the present invention. Among them: 1. Monochromatic illumination light source; 2. Optical pinhole; 3. Lens; 4. Beam splitter; 5. Phase modulator; 6. Microsphere lens; 7. Imaging sample; 8. Three-dimensional displacement stage; 9. Photomultiplier tube. The light emitted by the monochromatic illumination light source passes through the optical pinhole located on the focal plane of the lens and is then collimated into parallel light by the lens, and then reflected by the beam splitter to the phase modulator. Through the phase modulation of the incident light field by the phase modulator, a finer focused spot can be obtained on the lower microsphere lens. Scanning the surface of the sample with this focal spot, the scattered light on the surface enters the photomultiplier tube after passing through the microsphere lens and the lens, and a super-resolution imaging result can be obtained.

[0016] Figure 2 Schematic diagram of the structure of the phase modulator involved in the present invention. The phase modulator is composed of a substrate and a phase modulation unit. Among them, the substrate is a transparent dielectric material with a flat surface, which plays a supporting role, while the phase modulation unit can play a role in phase modulation. Different phase modulation units are made of the same material but have different thicknesses. In order to make the transmitted light beams of two adjacent modulation units have a phase difference of π, the thickness difference between them can be Δ h = λ / 2( n -1), where λ is n is the refractive index of the material of the phase modulation unit. If a modulation unit is arranged on each side of the central phase modulation unit, a one-dimensional phase modulator is formed, as shown in Figure 2 (b); if four phase modulators are arranged in two directions, the central focused spot can be compressed in two directions to form a two-dimensional phase modulator, as shown in Figure 2 (c).

[0017] To explain the compression effect of the phase modulation involved in the present invention on the focused spot, Figure 3 , Figure 4 and Figure 5 simulated the focusing effects of the microsphere lens in different situations. In Figure 3 , we simulated the focusing effect of the microsphere lens without a phase modulator. Set the incident wavelength to 500 nm, and select a SiO 2 microsphere with a diameter of 4 μm. Set the brightest position at the center of its focal spot as the focal plane, then the light intensity distribution on the focal plane is as shown in Figure 3 (a), the central intensity is very high, and there is a circle of very low-brightness side lobes around it. Along the position of the white dotted line in the figure, the light intensity distribution curve can be drawn, as shown in Figure 3As shown in (b). According to this light intensity curve, we can calculate the full width at half maximum (FWHM) of this focused spot. When a plane wave is incident and without any modulation, its FWHM is 320 nm, and this value is already better than that of an ordinary microscope objective lens with a numerical aperture of 0.9.

[0018] In Figure 4 , we keep the above parameters unchanged and add a one-dimensional phase modulator above the microsphere lens. Its structure is as shown in Figure 2 (b). There is a phase modulation unit on each side of the central phase modulation unit to simulate the influence of phase modulation on the focusing effect of the microsphere lens. The substrate is set as a SiO 2 flat plate with a thickness of 100 μm, and the material of the phase modulation unit is set as CeO with a refractive index of 2.35 2 . The thickness of the central unit is 100 nm, while the thickness of the phase modulation units on both sides is 285 nm. The diameter of each unit is 500 nm, and the interval is 300 nm. At this time, the focused spot as shown in Figure 4 (a) can be obtained on the focal plane of the microsphere lens. It can be seen from the figure that three circular units form three focused spots, which are close to each other but not connected, and the central spot is squeezed and even elongated by the spots on both sides. Along the white dotted line, its central light intensity distribution can be drawn, as shown in Figure 4 (b). The intensity of the central spot is weak, but its FWHM is reduced to about 210 nm after being compressed, which has been greatly improved compared with the focusing ability of the microsphere lens itself.

[0019] In Figure 5 , we replace the one-dimensional phase modulator with a two-dimensional phase modulator, that is, use the two-dimensional phase modulator shown in Figure 2 (c). There are four adjacent phase modulation units outside the central unit, and their respective parameters are the same as those used in Figure 4 . At this time, the focusing effect of the microsphere lens obtained is as shown in Figure 5 (a). Under the squeezing of the four-direction spots, the central spot forms a bright spot in the center, while the four focal spots around interfere with each other to form darker spots, and they are not connected to each other. Along the white dotted line, its central light intensity distribution is drawn, as shown in Figure 5 (b). The intensity of the central spot is the strongest, and its FWHM remains at about 220 nm after being squeezed in two directions, which has been greatly improved compared with the focusing ability of the microsphere lens itself. Applying this focused spot to confocal scanning imaging can achieve an imaging resolution within 100 nm.

[0020] A superfocusing microsphere lens and a super-resolution imaging system based on phase modulation, which combines a microsphere lens and a confocal imaging system, and proposes a method that can further compress the focal spot size of the microsphere lens and improve the imaging resolution of the confocal system by utilizing the influence of the incident light field phase on the focusing performance of the microsphere lens. Compared with the existing microsphere-assisted super-resolution imaging system and confocal imaging system, this solution can achieve an imaging resolution of 100 nm or even 50 nm, and has a broad application space. Compared with other super-resolution imaging technologies, the super-resolution imaging technology involved in the present invention does not require fluorescence staining, does not require image post-processing, and has strong compatibility with confocal optical microscopes. Therefore, it can be applied to the rapid imaging measurement of high-precision and large-area industrial samples.

[0021] The ultra-fine focusing microsphere lens involved in the above case is only an example, and the present invention includes but is not limited to the above microsphere lens structure. The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A super-focusing microsphere lens and super-resolution imaging system based on phase modulation, characterized in that The system consists of a monochromatic illumination source, an optical pinhole, a lens, a beam splitter, a phase modulator, a microsphere lens, an imaging sample, a three-dimensional translation stage, and a photomultiplier tube. Compared with the common microsphere-assisted super-resolution imaging system, the present invention sets a phase modulator above the microsphere lens, and by adjusting the phase of the incident light, the focal spot size of the microsphere lens can be compressed, and the imaging resolution of the confocal system can be improved.

2. The monochromatic illumination light source according to claim 1 usually has a spectrum width less than 10 nm. Commonly used light sources may be He-Ne lasers, solid lasers, semiconductor lasers, etc.

3. The phase modulator according to claim 1, characterized in that It consists of a substrate and a protruding circular phase modulation unit. Both the substrate and the phase modulation unit are made of transparent dielectric materials. The thickness of the substrate is in the order of hundreds of micrometers to millimeters.

4. The phase modulator according to claim 1, wherein the thickness of the protruding circular phase modulation unit is in the order of hundreds of nanometers, and the refractive index is n , the thickness difference between two adjacent circular phase modulation units is h , and satisfies h ( n -1) = λ / 2, where λ is the wavelength of monochromatic illumination light.

5. The phase modulator according to claim 1, wherein the diameter of the circular phase modulation unit is about several hundred nanometers.

6. The microsphere lens according to claim 1 is a dielectric microsphere with a diameter of about 2 to 9 microns.

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