A prism-based light source integrated super-resolution microscopy chip and its imaging method

Abstract

The present invention discloses a prism-based light source integrated super-resolution microscopy chip and an imaging method thereof, relating to the field of super-resolution microscopy. Based on a bulk high-refractive-index substrate material, a sample area is provided at the center of its upper surface, and multiple circles of prisms with different angles are provided on its lower surface. LEDs are respectively placed on the sides of the prisms, and then the positive and negative electrodes of the LEDs are integrated onto a PCB board, and the on / off of the LEDs is controlled by a single-chip microcomputer. Compared with the super-resolution microscopy imaging system with an external optical path, integrating the light source onto the super-resolution microscopy chip can achieve miniaturization and portability of super-resolution imaging, which is convenient for medical diagnosis and scientific research analysis in resource-scarce areas. Compared with traditional Fourier ptychography microscopy (FPM), using prisms can increase the light intensity, signal-to-noise ratio and oblique incidence range of the obliquely incident light source, introduce evanescent waves with a larger transverse wave vector, and effectively improve the imaging quality and resolution.

Description

Technical Field

[0001] The present invention relates to the field of super-resolution microscopy, and particularly to the field of on-chip frequency-shifted super-resolution microscopy imaging. Background Art

[0002] Traditional optical microscopy technology has played a huge role in the fields of biology, medicine, materials science, etc. However, due to the limitation of the Abbe diffraction limit, its imaging performance still cannot meet the high-precision and large-range detection requirements of these fields. In order to break through the diffraction limit of the optical system, a series of super-resolution microscopy imaging technologies have been invented. Among them, the super-resolution microscopy imaging technology based on spatial frequency shift can break through the bandwidth limitation of traditional imaging and detection devices, and has significant advantages in terms of large field of view and high speed.

[0003] The super-resolution microscopy imaging technology based on spatial frequency shift captures a series of low-resolution images, moves the information carrying spatial high frequencies into the spatial frequency domain that can be collected by the numerical aperture (NA) of the objective lens, and then performs algorithmic stitching in the frequency domain to restore a high-resolution image.

[0004] Existing frequency-shifted super-resolution microscopy imaging technologies include GaP-on-SiO 2 Integrated optical waveguide type super-resolution microscopy technology and wafer type super-resolution microscopy technology, etc. Although these technologies can achieve very high resolutions, their field of view ranges are all below the millimeter level and cannot meet the large field of view imaging requirements such as observing the large activity range of living tissues. In addition, the above technologies all require an external optical path to achieve illumination of the sample, and cannot be miniaturized and portable, and are not suitable for medical diagnosis and scientific research analysis in resource-scarce areas.

[0005] Some other frequency-shifted super-resolution microscopy imaging technologies, such as Fourier ptychography microscopy (FPM), use a series of LEDs with different distances and directions for illumination to capture corresponding low-resolution images, which can achieve miniaturization of the entire system to a certain extent. However, the illumination method of traditional FPM is that the LED is incident on the sample area through air, and its maximum transverse wave vector is limited by the air refractive index, so the highest resolution that can be achieved is limited. And traditional FPM uses a vertical illumination LED, and the light intensity and signal-to-noise ratio of the obliquely incident light source will be greatly reduced. Summary of the Invention

[0006] The object of the present invention is to propose a prism-based light source integrated super-resolution microscopy chip and its imaging method in view of the deficiencies of the prior art. The present invention mainly improves the efficiency of the LED obliquely incident on the sample area and the transverse wave vector of illumination through the prism coupling method, thereby improving its imaging quality and resolution.

[0007] The specific technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a prism-based light source integrated super-resolution microscopy chip, including a base material and a plurality of prisms; the base material has two flat and mutually parallel functional surfaces and an imaging surface, the center of the imaging surface is used to place the sample and serves as the sample area; on the functional surface, with the sample area as the center, a plurality of circles of prisms at different angles are coaxially arranged to improve the efficiency of the light source obliquely incident on the sample area and the lateral wave vector of the illumination.

[0009] Preferably, all areas on the imaging surface except the sample area are subjected to light shielding treatment, and all areas on the functional surface except where the prism is located are subjected to light shielding treatment to improve the signal-to-noise ratio of the image.

[0010] Furthermore, the shading treatment adopts micro-nano processing technology such as magnetron sputtering coating, and the shading material is selected from one of the materials with large light loss such as Cr, Au, Ag, etc. The thickness of the shading material layer should be the thinnest thickness while ensuring the shading effect.

[0011] As a preference, if the prism can be processed, try to choose a material with a high refractive index and low light loss in the optical band used, such as TiO 2 、SiO 2 、Al 2 O 3 、Si 3 N 4 , K9 glass. The higher the refractive index, the greater the frequency shift. The specific selection needs to be based on cost, processing conditions, etc.

[0012] Preferably, the prism is bonded to the base material using a transparent ultraviolet gluing method.

[0013] Preferably, in order to improve the efficiency of LED incident on the sample area, the surfaces of the prism that need to be polished include: the contact surface with the base material and the contact surface with the light source.

[0014] Preferably, the substrate material is a block or sheet structure. The cross section can be any shape as long as the prisms are symmetrically distributed around the sample.

[0015] In a second aspect, the present invention provides an imaging method using any of the prism-based light source integrated super-resolution microscopy chips described in the first aspect, as follows:

[0016] S1: Place the sample in the sample area, fix LEDs (such as SMD LEDs and plug-in LEDs) on the side of each prism away from the sample area as light sources, integrate all LEDs into the control circuit, and then place the whole on the sample stage of an ordinary optical microscope; the incident angle θ of the mth circle LED as the light source is obliquely incident on the center of the sample mSatisfy the following formula:

[0017]

[0018] In the formula, the thickness of the base material is T, the total number of prism circles distributed on the functional surface is n, and the linear distance between the center of the m-th prism circle and the sample center is L m , n is an integer and 1 ≤ m ≤ n;

[0019] S2: Illuminate the sample through an ordinary optical microscope, and collect the low-frequency spatial information of the sample with an optical camera;

[0020] S3: Light up the LEDs on each circle of prisms in sequence from the inside to the outside, so that the light source obliquely enters the sample area to illuminate the sample, and collect the high-frequency light field information of different frequencies of the sample under illumination by light sources from different circles and different directions with an optical camera;

[0021] S4: Based on the collected low-frequency spatial information and high-frequency light field information, perform iterative stitching in the frequency domain space to obtain an enlarged spectrum, and then perform inverse Fourier transform to the spatial domain, that is, reconstruct the super-resolution image of the sample.

[0022] The wave vector that the obliquely incident light can provide is: K m = K 0 *N m , where K 0 is the wave vector of the incident light in vacuum, which can be expressed as λ is the wavelength of the incident light; N m = N*cosθ m is the effective refractive index of the material, and N is the refractive index of the base material. It can be seen from the above formula that the higher the effective refractive index, the greater the frequency shift amount. Generally speaking, the number of prism circles is designed according to the required frequency shift amount. The greater the frequency shift amount, the more prism circles. The size of the prism depends on the size of the LED and the size of the base material. In order to ensure the quality of the reconstructed image, the corresponding frequency-shifted spectra of each prism should have a certain overlap rate. Therefore, generally speaking, on the premise that the prisms can be distributed on the base, the number of inner-circle prisms should be designed to be less, and the number of outer-circle prisms should be designed to be more.

[0023] The effective imaging field of view (FOV) (i.e., the sample area) is the overlapping area where the LED is incident on the imaging surface through each prism.

[0024] Preferably, the LED is fixedly adhered to the prism through a transparent ultraviolet glue.

[0025] Preferably, the positive and negative poles of the LED are integrated onto a PCB board, and the on-off control of each LED light source is realized through single-chip microcomputer programming.

[0026] The present invention has the following beneficial effects compared with the prior art:

[0027] In the present invention, the LED light source is integrated onto the super-resolution microscopy chip, eliminating the need for an external optical path, which is conducive to the miniaturization and portability of super-resolution microscopy imaging. Secondly, in the present invention, a prism is used to obliquely incident the LED light source onto the sample area. Compared with the traditional FPM, the light intensity and signal-to-noise ratio of each LED light source obliquely incident onto the sample area can be increased, improving the imaging quality; due to the adoption of a new illumination structure, an evanescent wave with a larger transverse wave vector is introduced, which can further improve the imaging resolution. Description of the Drawings

[0028] Figure 1 is a schematic cross-sectional view of the chip;

[0029] Figure 2 is the top view (a) and the front view (b) of the prism;

[0030] Figure 3 is a planar distribution diagram of the prism in the embodiment;

[0031] Figure 4 is a schematic diagram of the reconstructed spectrum in the embodiment. Detailed Embodiments

[0032] The present invention will be further described and illustrated below in conjunction with the drawings and specific embodiments. The technical features of each embodiment in the present invention can be combined correspondingly without conflict.

[0033] Embodiment

[0034] Figure 1 is a schematic cross-sectional view of the chip of the present invention, with a thickness of T and a diameter of D. The upper surface of the chip is the imaging surface, the center is the sample area, and the rest is coated with a chromium film with a thickness of 300 nm as a light-shielding layer. The lower surface of the chip is the functional surface, with 9 circles of prisms at different angles distributed, and the rest is also coated with a chromium film with a thickness of 300 nm as a light-shielding layer. Figure 2 is the top view (a) and the front view (b) of the prism. The triangular side of each prism is a right triangle, and the θ m angle is the angle of the light source obliquely incident onto the sample area. The side length of the prism in contact with the chip on the triangular side is b, the side length in contact with the LED is a, and the other side is c. The top view of the prism is a rectangle, with two side lengths of b and the other two side lengths of d. Figure 3 is a planar schematic diagram of the prism arrangement in this embodiment. The total number of circles of the prism n is set to 9, and the lateral distance from the center of the m-th circle of the prism to the center of the sample is L m .

[0035] When the LED in the m-th circle obliquely irradiates the center of the sample through the prism attached to it, the incident angle is θm :

[0036]

[0037] The wave vector provided by the obliquely incident light is: K m = K 0 *N m , where K 0 is the wave vector of the incident light in vacuum and can be expressed as λ is the wavelength of the incident light; N m = N*cosθ m is the effective refractive index of the material, and N is the refractive index of the substrate material.

[0038] The following are the specific parameters of this embodiment:

[0039] Objective lens: The magnification is selected to be 5 times, and its NA is 0.12;

[0040] LED: The wavelength is 460 nm and the diameter is 3.5 mm;

[0041] Substrate material: The refractive index is 1.53, T = 50 mm, D = 150 mm;

[0042] Imaging field of view: 9.68 mm 2 .

[0043] The specific parameters of the prism and the effective refractive index per turn are shown in Table 1.

[0044] The reconstructed spectral space of the sample obtained by the chip of the present invention is as Figure 4 shown. It can be seen that the spectral space is significantly expanded compared with that of an ordinary microscope. Theoretically, its resolution can reach 338 nm, and the resolution can be improved by 11 times compared with an ordinary microscope with NA = 0.12 / ×5; the synthetic NA can reach 1.36, which is much higher than the synthetic NA (0.3NA - 0.7NA) of the traditional FPM.

[0045] Table 1 Specific parameters of the prism and the effective refractive index per turn

[0046]

[0047]

[0048] Compared with the super-resolution microscopy imaging system with an external optical path, integrating the light source onto the super-resolution microscopy chip can achieve miniaturization and portability of super-resolution imaging, facilitating its application in medical diagnosis and scientific research analysis in resource-scarce areas. Compared with traditional Fourier ptychography microscopy (FPM), using a prism can increase the light intensity, signal-to-noise ratio, and oblique incidence range of the obliquely incident light source, introduce evanescent waves with a larger transverse wave vector, and effectively improve the imaging quality and resolution.

[0049] The above-described embodiments are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent replacement or equivalent transformation methods fall within the protection scope of the present invention.

Claims

1. A prism-based light source integrated super-resolution microscopy chip, characterized in that, it includes a substrate material and a number of prisms; the substrate material has two flat and parallel functional surfaces and an imaging surface, and the center of the imaging surface is used to place a sample and serves as the sample area; on the functional surface, centered on the sample area, a number of circles of prisms with different angles are coaxially arranged to improve the efficiency of oblique incidence of the light source into the sample area and the lateral wave vector of illumination; the other areas on the imaging surface except the sample area are shaded, and the other areas on the functional surface except where the prisms are located are shaded; the shading treatment is achieved through a shading material, and the shading material is selected from one of Cr, Au, and Ag materials, and the thickness of the shading material layer is selected to be the thinnest on the premise of ensuring the shading effect; An LED is fixed on the side of each prism away from the sample area as a light source, and all the LEDs are integrated into a control circuit; the triangular side of each prism is a right triangle, the side length in contact with the functional surface of the base material on the right triangle is b, the side length in contact with the LED is a, and the other side is c; the top projection of the prism is a rectangle, and the side lengths of the rectangle are b and d respectively; the incident angle θ of the m-th circle of LEDs as light sources obliquely incident into the center of the sample m Satisfies the following formula: Wherein, the thickness of the base material is T, the total number of prism turns distributed on the functional surface is n, and the linear distance between the center of the m-th prism turn and the center of the sample is L m , n is an integer and 1 ≤ m ≤ n.

2. The prism-based light source integrated super-resolution microscopy chip according to claim 1, characterized in that, The prism material is TiO 2 , SiO 2 , Al 2 O 3 , Si 3 N 4 , or one of K9 glasses.

3. The prism-based light source integrated super-resolution microscopy chip according to claim 1, characterized in that, the combination of the prism and the substrate material adopts a transparent ultraviolet glue bonding method.

4. The prism-based light source integrated super-resolution microscopy chip according to claim 1, characterized in that, the contact surfaces of the prism with the substrate material and with the light source are polished.

5. The prism-based light source integrated super-resolution microscopy chip according to claim 1, characterized in that, the substrate material is in a block or sheet structure.

6. An imaging method using the prism-based light source integrated super-resolution microscopy chip according to any one of claims 1 to 5, characterized in that, specifically as follows: S1: Place the sample in the sample area. Fix LEDs as light sources on the sides of each prism away from the sample area respectively, then integrate all the LEDs into the control circuit, and subsequently place the whole on the sample stage of an ordinary optical microscope. Among them, the incident angle θ of the m-th ring of LEDs as light sources obliquely incident into the center of the sample m satisfies the following formula: Wherein, the thickness of the base material is T, the total number of prism rings distributed on the functional surface is n, and the linear distance between the center of the m-th prism ring and the center of the sample is L m , n is an integer and 1 ≤ m ≤ n; S2: Illuminate the sample through an ordinary optical microscope, and use an optical camera to collect the low-frequency spatial information of the sample; S3: Sequentially light up the LEDs on each circle of prisms from the inside to the outside, so that the light source obliquely enters the sample area to illuminate the sample, and use an optical camera to collect the high-frequency light field information of different frequencies of the sample illuminated by light sources from different circles and different directions; S4: Based on the collected low-frequency spatial information and high-frequency light field information, perform iterative stitching in the frequency domain space to obtain an enlarged spectrum, and then perform inverse Fourier transform to the spatial domain, that is, reconstruct the super-resolution image of the sample.

7. The imaging method according to claim 6, characterized in that, the LED is fixed to the prism by transparent ultraviolet glue.

8. The imaging method according to claim 6, characterized in that, the positive and negative poles of the LED are integrated onto a PCB board, and the on / off control of each LED light source is realized through single-chip microcomputer programming.

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