阅读说明：本技术 衍射层析显微成像系统及方法 (Diffraction chromatography microscopic imaging system and method ) 是由 吴芹芹 黄伟 于 2020-06-04 设计创作，主要内容包括：本发明公开了衍射层析显微成像系统,包括：承载片,包括透明载片和反射膜,透明载片包括相对的第一表面和第二表面,第一表面用于承载样品,反射膜设置于第二表面上；光路组件,用于产生参考光以及照射样品的入射光,并对参考光、第一样品光和第二样品光进行合束；显微镜组件,用于使入射光通过而照射到样品背向第一表面的前表面和第一表面上,并且使第一样品光和第二样品光通过；成像组件,用于采集合束后的参考光、第一样品光和第二样品光的干涉图像。本发明还公开了采用了上述成像系统的衍射层析显微成像方法。本发明解决了在衍射层析术中,入射光只能扫描到样品朝向物镜的一侧表面,而无法扫描到样品背向物镜的一侧表面的问题。(The invention discloses a diffraction chromatography microscopic imaging system, comprising: the carrier sheet comprises a transparent carrier sheet and a reflecting film, the transparent carrier sheet comprises a first surface and a second surface which are opposite, the first surface is used for carrying a sample, and the reflecting film is arranged on the second surface; the light path component is used for generating reference light and incident light for irradiating the sample, and combining the reference light, the first sample light and the second sample light; a microscope assembly for passing incident light to irradiate onto a front surface of the sample opposite to the first surface and the first surface, and passing the first sample light and the second sample light; and the imaging component is used for acquiring the interference images of the combined reference light, the first sample light and the second sample light. The invention also discloses a diffraction chromatography microimaging method adopting the imaging system. The invention solves the problem that in diffraction chromatography, incident light can only scan the surface of one side of a sample facing to an objective lens, but can not scan the surface of one side of the sample opposite to the objective lens.)

1. A diffraction tomography microscopic imaging system, comprising:

the carrier sheet comprises a transparent carrier sheet and a reflecting film, wherein the transparent carrier sheet comprises a first surface and a second surface which are opposite, the first surface is used for carrying a sample, and the reflecting film is arranged on the second surface;

the light path component is used for generating reference light and incident light for irradiating the sample, and combining the reference light, the first sample light and the second sample light;

a microscope assembly for passing the incident light to irradiate on a front surface of the sample facing away from the first surface and the first surface, and passing a first sample light and a second sample light;

the imaging assembly is used for acquiring interference images of the reference light, the first sample light and the second sample light after beam collection;

wherein the incident light generates the first sample light by reflecting on and/or passing through a front surface of the sample when the incident light is irradiated to the front surface of the sample;

the incident light is irradiated to the first surface, and when reflected on the reflective film to be irradiated to the back surface of the sample, the incident light is reflected on the back surface of the sample and/or passes through the back surface of the sample to form the second sample light;

the first sample light generated by reflection on the front surface of the sample and/or the second sample light generated by passing through the back surface of the sample are directed towards the microscope assembly; the first sample light generated through the front surface of the sample and/or the second sample light generated by reflection on the back surface of the sample is directed towards the microscope assembly after reflection on the reflective film.

2. The diffractive tomography microscopic imaging system of claim 1, wherein said optical path assembly comprises:

a light source for generating original light;

a beam splitter for splitting the original light to form the reference light and the incident light;

a beam combiner for combining the reference light, the first sample light and the second sample light.

3. The diffractive tomography microscopic imaging system of claim 2, wherein said optical path assembly further comprises:

the first collimation beam expander is used for collimating and expanding the incident light;

the galvanometer is used for adjusting the incident angle of the incident light so that the incident light is incident to the microscope component through the beam combiner at different incident angles;

the second collimation beam expander is used for collimating and expanding the reference light;

and the reflector component is used for reflecting the collimated and expanded reference light to the beam combiner.

4. The diffraction tomography microscopy imaging system of claim 3, wherein the mirror assembly comprises: a first mirror and a second mirror;

the first reflector is used for reflecting the reference light after collimation and beam expansion to the second reflector, and the second reflector is used for reflecting the reference light reflected to the second reflector by the first reflector to the beam combiner.

5. The diffraction tomography microscopy imaging system of claim 1, wherein the imaging assembly comprises: a polarizer and a camera;

wherein the combined reference light, the first sample light and the second sample light reach the camera after passing through the polarizer.

6. The diffraction tomography microscopy imaging system as claimed in claim 1, wherein the microscope assembly comprises a lens and an objective lens, the incident light passing through the lens and the objective lens in sequence and impinging on a front surface of the sample facing away from the first surface and the first surface; wherein the first sample light and the second sample light sequentially pass through the objective lens and the lens and then face the optical path component.

7. A diffraction tomographic microscopic imaging method using the diffraction tomographic microscopic imaging system according to any one of claims 1 to 6, the method comprising:

carrying a sample on a first surface of a transparent slide of the carrying sheet, wherein a reflecting film is arranged on a second surface of the transparent slide opposite to the first surface;

generating reference light and incident light for irradiating the sample by using the light path component;

irradiating the incident light onto a front surface of the sample opposite to the first surface and a back surface facing the first surface through a microscope assembly and the reflective film to generate first and second sample lights;

combining the first sample light and the second sample light with the reference light in the light path component after passing through the microscope component;

acquiring interference images of the combined reference light, the first sample light and the second sample light by using an imaging assembly;

wherein the incident light generates the first sample light by reflecting on and/or passing through a front surface of the sample when the incident light is irradiated to the front surface of the sample;

the incident light is irradiated to the first surface, and when reflected on the reflective film to be irradiated to the back surface of the sample, the incident light is reflected on the back surface of the sample and/or passes through the back surface of the sample to form the second sample light;

the first sample light generated by reflection on the front surface of the sample and/or the second sample light generated by passing through the back surface of the sample are directed toward the optical path component; the first sample light generated through the front surface of the sample and/or the second sample light generated by reflection on the back surface of the sample are directed toward the light path component after reflection on the reflective film.

Technical Field

The invention belongs to the technical field of microscopic imaging, and particularly relates to a diffraction tomography microscopic imaging system and method which can realize near isotropy of resolution without rotating a sample while tomography is carried out by adopting a light source.

Background

The chromatographic microscopic imaging technology is a technical means for effectively obtaining the fault information of a tiny sample and realizing the three-dimensional measurement of the internal structure of the sample. Typical tomographic microscopy techniques include X-ray computer tomography, laser scanning confocal microscopy, diffraction tomographic microscopy, and the like. The imaging technologies are widely applied in the fields of medical science, micro-optical device detection and the like, so that the development of the technologies of medical science detection, micro-nano processing and the like is greatly promoted.

The diffraction chromatography microimaging technology is as follows: and acquiring the scattered field distribution of the sample at each angle by using various interference technologies, and reconstructing the refractive index three-dimensional distribution by using a Fourier diffraction chromatography algorithm. The diffraction tomography microscopic imaging technology can carry out real-time, undisturbed, dynamic and quantitative imaging on the sample. Among the diffraction tomographic microscopic imaging techniques, there are a transmission diffraction tomographic microscopic imaging technique and a reflection diffraction tomographic microscopic imaging technique according to the type of a sample.

In the diffraction tomography microscopy, a light source is used for tomography, and since the slide carrying the sample is transparent, the original light only impinges on the surface of the sample facing the objective lens (i.e. facing away from the slide), whereas the original light not impinging on the sample passes directly through the slide and thus does not impinge on the surface of the sample facing away from the objective lens (i.e. in contact with the slide). Therefore, the simple diffraction tomography microscopic imaging technique cannot obtain refractive index distribution with isotropic resolution. To solve such a problem, a method generally adopted in the prior art is to scan one side of a sample and then rotate the sample, thereby scanning the other side of the sample, so that the scattered field distribution of a large viewing angle can be recorded to realize near isotropy of resolution in a three-dimensional refractive index map. However, this method causes the spectrum loss of the shape of the apple kernel in the EWald frequency domain sphere, i.e. there is a problem of "apple kernel spectrum loss", and the parasitic motion accompanied by the mechanical rotation of the sample and the deformation of the sample during the rotation greatly reduce the accuracy of image reconstruction.

Disclosure of Invention

In order to solve the technical problems, the invention adopts the following technical scheme:

in one aspect of the invention there is provided a diffraction tomography microscopy imaging system, the system comprising:

the carrier sheet comprises a transparent carrier sheet and a reflecting film, wherein the transparent carrier sheet comprises a first surface and a second surface which are opposite, the first surface is used for carrying a sample, and the reflecting film is arranged on the second surface;

the light path component is used for generating reference light and incident light for irradiating the sample, and combining the reference light, the first sample light and the second sample light;

a microscope assembly for passing the incident light to irradiate on a front surface of the sample facing away from the first surface and the first surface, and passing a first sample light and a second sample light;

the imaging assembly is used for acquiring interference images of the reference light, the first sample light and the second sample light after beam collection;

wherein the incident light generates the first sample light by reflecting on and/or passing through a front surface of the sample when the incident light is irradiated to the front surface of the sample;

the incident light is irradiated to the first surface, and when reflected on the reflective film to be irradiated to the back surface of the sample, the incident light is reflected on the back surface of the sample and/or passes through the back surface of the sample to form the second sample light;

the first sample light generated by reflection on the front surface of the sample and/or the second sample light generated by passing through the back surface of the sample are directed towards the microscope assembly; the first sample light generated through the front surface of the sample and/or the second sample light generated by reflection on the back surface of the sample is directed towards the microscope assembly after reflection on the reflective film.

Preferably, the optical path component includes:

a light source for generating original light;

a beam splitter for splitting the original light to form the reference light and the incident light;

a beam combiner for combining the reference light, the first sample light and the second sample light.

Preferably, the optical path component further includes:

the first collimation beam expander is used for collimating and expanding the incident light;

the galvanometer is used for adjusting the incident angle of the incident light so that the incident light is incident to the microscope component through the beam combiner at different incident angles;

the second collimation beam expander is used for carrying out collimation and beam expansion on the reference light;

and the reflector component is used for reflecting the collimated and expanded reference light to the beam combiner.

Preferably, the mirror assembly comprises: a first mirror and a second mirror;

the first reflector is used for reflecting the reference light after collimation and beam expansion to the second reflector, and the second reflector is used for reflecting the reference light reflected to the second reflector by the first reflector to the beam combiner.

Preferably, the imaging assembly comprises: a polarizer and a camera;

wherein the combined reference light, the first sample light and the second sample light reach the camera after passing through the polarizer.

Preferably, the microscope component comprises a lens and an objective lens, and the incident light is irradiated on the front surface of the sample opposite to the first surface and the first surface after sequentially passing through the lens and the objective lens; wherein the first sample light and the second sample light sequentially pass through the objective lens and the lens and then face the optical path component.

In another aspect of the present invention, there is provided a diffraction tomography microscopic imaging method using the diffraction tomography microscopic imaging system as described above, the method comprising:

carrying a sample on a first surface of a transparent slide of the carrying sheet, wherein a reflecting film is arranged on a second surface of the transparent slide opposite to the first surface;

generating reference light and incident light for irradiating the sample by using the light path component;

irradiating the incident light onto a front surface of the sample opposite to the first surface and a back surface facing the first surface through a microscope assembly and the reflective film to generate first and second sample lights;

combining the first sample light and the second sample light with the reference light in the light path component after passing through the microscope component;

acquiring interference images of the combined reference light, the first sample light and the second sample light by using an imaging assembly;

wherein the incident light generates the first sample light by reflecting on and/or passing through a front surface of the sample when the incident light is irradiated to the front surface of the sample;

the incident light is irradiated to the first surface, and when reflected on the reflective film to be irradiated to the back surface of the sample, the incident light is reflected on the back surface of the sample and/or passes through the back surface of the sample to form the second sample light;

the first sample light generated by reflection on the front surface of the sample and/or the second sample light generated by passing through the back surface of the sample are directed toward the optical path component; the first sample light generated through the front surface of the sample and/or the second sample light generated by reflection on the back surface of the sample are directed toward the light path component after reflection on the reflective film.

The diffraction tomography micro-imaging system provided by the invention is provided with the reflecting film on the bearing sheet for bearing the sample, the reflecting film is adopted to change the optical path of part of the incident light irradiated outside the sample, so that the incident light can be irradiated to the back surface of the sample (namely, the side surface of the sample facing the bearing surface of the bearing sheet), thereby the diffraction tomography micro-imaging system provided by the invention can scan the back surface of the sample to generate the second sample light with the back surface information of the sample, namely, the diffraction tomography micro-imaging system provided by the invention can not only generate the first sample light with the front surface information of the sample and generated by irradiating the incident light on the front surface of the sample, but also generate the second sample light with the back surface information of the sample, thereby realizing that the scattered field distribution with large visual angle can be recorded without rotating the sample, near isotropy of the resolution of the three-dimensional refractive index map is achieved.

Furthermore, in the diffraction tomography micro-imaging system provided by the invention, the bearing sheet for bearing the sample is provided with the reflecting film, and the incident light can also pass through the front surface of the sample to form the first sample light which is reflected to the light path component, so that the diffraction tomography micro-imaging system provided by the invention can be compatible with the reflective sample, the transmissive sample and the mixed sample at the same time, and the universality of the diffraction tomography micro-imaging system is improved.

Drawings

FIG. 1 is an optical path diagram of a diffraction tomography microscopy imaging system according to an embodiment of the present invention;

FIG. 2 is a diagram of an optical path for scanning a reflective sample in an embodiment of the present invention;

FIG. 3 is a diagram of an optical path for scanning a transmissive sample in an embodiment of the present invention;

FIG. 4 is a diagram of an optical path for scanning a mixed sample in accordance with an embodiment of the present invention;

FIG. 5 is a flow chart of a diffraction tomography microscopic imaging method according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described in accordance with the drawings are exemplary only, and the invention is not limited to these embodiments.

It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the scheme according to the present invention are shown in the drawings, and other details not so relevant to the present invention are omitted.

Example 1

The present embodiment provides a diffraction tomography microscopic imaging system, as shown in fig. 1, comprising: the microscope comprises a bearing sheet 1, an optical path component 2, a microscope component 3 and an imaging component 4.

As shown in fig. 2, the carrier sheet 1 includes a transparent carrier sheet 11 and a reflective film 12, the transparent carrier sheet 11 includes a first surface 11a and a second surface 11B opposite to each other, the first surface 11a is used for carrying a sample B, and the reflective film 12 is disposed on the second surface 11B. The material of the reflective film 12 may be aluminum or silver, and the transparent carrier 11 is preferably made of a transparent polymer material.

The optical path component 2 is used for generating reference light A2 and incident light A1 for irradiating the sample B. Specifically, the optical path component 2 includes: the device comprises a light source 21, a beam splitter 22, a first collimation beam expander 24, a galvanometer 27, a second collimation beam expander 25, a reflector component and a beam combiner 23.

As shown in FIG. 1, the original light A passes through the beam splitter 22 to generate the incident light A1 and the reference light A2. The generated incident light a1 is collimated and spread by the first collimating and spreading beam device 24, then directed to the beam combiner 23 under the adjustment of the galvanometer 27, and enters the microscope assembly 3 through the beam combiner 23. The galvanometer 27 is configured to adjust the incident angle of the incident light a1, so that the incident light a1 is incident on the microscope assembly 3 via the beam combiner 23 at different incident angles, thereby realizing the illumination on the sample B at different angles, and obtaining information of different areas of the sample B.

The generated reference light a2 is collimated and expanded by the second collimating and expanding device 25, then reflected to the beam combiner 23 under the action of the mirror assembly, and enters the microscope assembly 3 through the beam combiner 23. Wherein the mirror assembly comprises: a first reflecting mirror 261 and a second reflecting mirror 262, wherein the first reflecting mirror 261 is used for reflecting the reference light A2 after collimation and expansion to the second reflecting mirror 262, and the second reflecting mirror 262 is used for reflecting the reference light A2 reflected by the first reflecting mirror 261 to the beam combiner 23.

A microscope assembly 3 for passing the incident light a1 to irradiate the front surface of the sample B facing away from the first surface 11a and the first surface 11a, so that the incident light a1 generates a first sample light B1 and a second sample light B2. The microscope assembly 3 comprises a lens 31 and an objective lens 32, and the incident light a1 is irradiated onto the front surface of the sample B opposite to the first surface 11a and the first surface 11a after passing through the lens 31 and the objective lens 32 in sequence.

The following describes a specific process of generating the first sample light B1 and the second sample light B2.

As shown in fig. 2, sample B is a reflective sample. In one aspect, when a portion of incident light a1 strikes the front surface of sample B (the side of sample B facing away from first surface 11a in fig. 2), the incident light a1 reflects off the front surface of sample B to generate the first sample light B1, and the generated first sample light B1 is directed toward the microscope assembly 3;

on the other hand, a part of the incident light a1 is irradiated onto the first surface 11a (i.e. not irradiated onto the sample itself), and is irradiated onto the back surface of the sample B by reflection of the reflective film 12 (the side of the sample B facing the first surface 11a in fig. 2), at which time the incident light a1 is reflected on the back surface of the sample B to form the second sample light B2, and the generated second sample light B2 is reflected on the reflective film 12 and then directed toward the microscope component 3 (in this process, the second sample light B2 may be reflected multiple times between the sample and the reflective film).

As shown in fig. 3, the sample B is a transmissive sample. In one aspect, a portion of the incident light a1 passes through the front surface of the sample B to generate the first sample light B1, the generated first sample light B1 being directed toward the microscope assembly 3 after reflection on the reflective film 12;

on the other hand, a part of the incident light a1 is irradiated onto the first surface 11a and irradiated onto the back surface of the sample B by reflection by the reflection film 12, and at this time, the incident light a1 passes through the back surface of the sample B to form the second sample light B2, and the second sample light B2 is generated to be directed toward the microscope assembly 3.

It should be noted here that the sample B may be a mixed sample. As shown in fig. 4, when the sample B is a mixed sample, the above two cases occur simultaneously, that is, when the incident light a1 is irradiated onto the front surface or the back surface of the sample B, the phenomenon that light is reflected on the surface of the sample and directly passes through the sample occurs simultaneously.

Further, the generated first sample light B1 and second sample light B2 sequentially pass through the objective lens 32 and the lens 31 and then face the beam combiner 23. The first sample light B1 and the second sample light B2 are captured by the imaging module 4 after being combined with the reference light a2 in the beam combiner 23.

Specifically, the imaging assembly 4 includes a polarizer 41 and a camera 42. The combined reference light a2, the first sample light B1, and the second sample light B2 reach the camera 42 after passing through the polarizer 41. The camera 42 captures the combined reference light a2, the first sample light B1 and the second sample light B2 to generate an interference image.

The diffraction tomography microscopic imaging system of the embodiment adopts the bearing sheet with the reflective film 12, so that when the light source 21 is adopted for tomography, light can be scanned to one side surface of the reflective sample B facing the objective lens 32, and can also be scanned to one side surface of the reflective sample B back to the objective lens 32 through the reflective film of the bearing sheet, so that the scattered field distribution of a large viewing angle can be recorded without rotating the reflective sample B, and the near isotropy of the resolution of the three-dimensional refractive index map can be achieved. Moreover, the diffraction tomography micro-imaging system of the embodiment can be compatible with the detection of the reflective sample, the transmissive sample and the mixed sample, so that the universality of the diffraction tomography micro-imaging system is improved.

Example 2

This example provides a diffraction tomography microscopic imaging method using the hardware of the diffraction tomography microscopic imaging system of example 1.

As shown in fig. 5, the method includes the following specific steps:

s1, carrying the sample B on a first surface 11a of a transparent slide 11 of the carrying sheet 1, wherein a reflecting film 12 is arranged on a second surface 11B of the transparent slide 11 opposite to the first surface 11 a;

s2, generating reference light A2 and incident light A1 for irradiating the sample B by using the optical path component 2;

s3, irradiating the incident light a1 through the microscope assembly 3 and the reflective film 12 onto the front surface of the sample B facing away from the first surface 11a and the back surface facing the first surface 11a to generate a first sample light B1 and a second sample light B2;

s4, combining the first sample light B1 and the second sample light B2 with the reference light A2 in the light path component 2 after passing through the microscope component 3;

s5, acquiring interference images of the combined reference light A2, the first sample light B1 and the second sample light B2 by using an imaging assembly 4;

wherein the incident light a1 reflects off of and/or passes through the front surface of the sample B to generate the first sample light B1 when the incident light a1 strikes the front surface of the sample B;

the incident light a1 is irradiated to the first surface 11a, and when reflected on the reflective film 12 to be irradiated to the back surface of the sample B, the incident light a1 is reflected on the back surface of the sample B and/or passes through the back surface of the sample B to form the second sample light B2;

the first sample light B1 generated by reflection on the front surface of the sample B and/or the second sample light B2 generated by passing through the back surface of the sample B is directed towards the light path assembly 2; the first sample light B1 generated through the front surface of the sample B and/or the second sample light B2 generated by reflection on the back surface of the sample B are directed toward the light path member 2 after reflection on the reflection film 12.

The sample B is one of a reflective sample, a transmissive sample, and a mixed sample.

In this document, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.