Phase contrast microscope, phase shift quantitative imaging method, system and storage medium

Abstract

The present application discloses a phase contrast microscope, a phase shift quantitative imaging method, a system and a storage medium. The microscope includes a dot matrix target ring light source, a pupil aperture, a sample stage, a zone plate objective lens, a dot matrix phase shift ring and a detector arranged in sequence. In the present application, the dot matrix target ring light source is imaged on the dot matrix phase shift ring by the zone plate objective lens, and each point source image has a uniquely corresponding phase filtering point on the dot matrix phase shift ring. Since the point source image and the phase filtering point are relatively small, after the sample is placed, most of the low-order diffracted light can pass unobstructed beside the phase filtering point, thereby maximizing the elimination of the cause of halo artifacts. Combining with a simple phase shift imaging method, the purpose of phase shift quantitative imaging is achieved.

Description

Technical Field

[0001] The present invention generally relates to the technical field of optical devices, and in particular to a phase contrast microscope, a phase shift quantitative imaging method, a system and a storage medium. Background Art

[0002] Living cells and unstained biological tissue samples, due to the different refractive index and thickness of the cell structure, will cause a small drop in light intensity and a large phase shift when light passes through. The phase shift produced by this sample is invisible to the human eye. Phase contrast microscopy changes the phase difference between zero-order transmitted light and diffracted light through a phase shift ring, and uses the interference of zero-order transmitted light and diffracted light to convert the phase shift produced by the sample into light intensity for imaging.

[0003] However, in traditional phase contrast microscopy, the phase shift ring blocks the effect of low-order diffracted light near the zero-order transmitted light, so that these low-order diffracted lights also introduce phase shifts, resulting in distortion and halo artifacts. At the same time, due to the different trends of the three functions of sample absorption, sample phase shift, and sine and cosine, it is not easy to solve the sample phase shift from the imaging light intensity formula. Therefore, there is currently no simple method for quantitative imaging of sample phase shift. Summary of the invention

[0004] In view of the above-mentioned defects or deficiencies in traditional phase contrast microscopes, it is desired to provide a new phase contrast microscope, phase shift quantitative imaging method, system and storage medium, which can not only effectively eliminate halo artifacts, but also easily perform quantitative imaging of sample phase shift.

[0005] In a first aspect, the present application provides a phase contrast microscope, which includes a lattice target ring light source, an aperture stop, a sample stage, a zone plate objective, a lattice phase shift ring, and a detector arranged in sequence.

[0006] Optionally, in some embodiments of the present application, the diameter of the phase filter point on the dot matrix phase shift ring is smaller than

[0007] Among them, S i represents the distance between the dot matrix phase shift ring and the zone plate objective lens, D represents the diameter of the zone plate objective lens, N represents the number of pixels corresponding to the field of view width of the object plane at the sample stage, and d o Indicates the distance between the object plane at the sample stage and the zone plate objective.

[0008] Optionally, in some embodiments of the present application, the diameter of the point source on the dot matrix target ring light source is smaller than

[0009] Among them, S o Indicates the distance between the dot matrix target ring light source and the zone plate objective.

[0010] Optionally, in some embodiments of the present application, the dot matrix target ring light source includes an X-ray source.

[0011] Optionally, in some embodiments of the present application, the phase shift of the lattice phase shift ring is

[0012] In a second aspect, the present application provides a phase shift quantitative imaging method, which is applied to the phase contrast microscope described in any one of the first aspects, comprising:

[0013] Adjusting the imaging optical path of the phase contrast microscope so that the center of the dot matrix target ring light source and the center of the dot matrix phase shift ring are located on the same optical axis, and the dot matrix target ring light source is imaged on the dot matrix phase shift ring through the zone plate objective lens, and each point source image is aligned with the corresponding phase filter point;

[0014] Respectively photographing a first sample image when the lattice phase shift ring is in place and a second sample image when the lattice phase shift ring is removed;

[0015] A sample phase shift image is calculated based on the first sample image and the second sample image.

[0016] Optionally, in some embodiments of the present application, the imaging light intensity of the first sample image is:

[0017]

[0018] Among them, γ represents the absorption attenuation of the lattice phase shift ring, and Φ represents the phase shift of the sample.

[0019] Optionally, in some embodiments of the present application, the imaging light intensity of the second sample image is:

[0020] I(X,Y)=exp[-M(X,Y)]

[0021] Where M represents the sample absorption.

[0022] Optionally, in some embodiments of the present application, the sample phase shift corresponding to the sample phase shift image is calculated by the following formula:

[0023]

[0024] Where γ = e -μt , μ and t represent the linear attenuation coefficient and thickness of the lattice phase shift ring, respectively.

[0025] In a third aspect, the present application provides a phase-shift quantitative imaging system, the system comprising a phase-shift quantitative imaging device and a phase contrast microscope according to any one of the first aspects, the phase-shift quantitative imaging device comprising:

[0026] An adjustment module, used for adjusting the imaging optical path of the phase contrast microscope, so that the center of the lattice target ring light source and the center of the lattice phase shift ring are located on the same optical axis, and the lattice target ring light source is imaged on the lattice phase shift ring through the zone plate objective lens, and each point source image is aligned with the corresponding phase filter point;

[0027] A shooting module, used for shooting a first sample image when the dot matrix phase shift ring is in place and a second sample image when the dot matrix phase shift ring is removed;

[0028] A calculation module is used to calculate a sample phase-shift image according to the first sample image and the second sample image.

[0029] In a fourth aspect, the present application provides a computer-readable storage medium, which stores one or more programs, and the one or more programs can be executed by one or more processors to implement the steps of the phase-shift quantitative imaging method described in any one of the second aspects.

[0030] In summary, the phase contrast microscope, phase shift quantitative imaging method, system and storage medium provided in the embodiments of the present application include a dot matrix target ring light source, an aperture, a sample stage, a zone plate objective lens, a dot matrix phase shift ring and a detector arranged in sequence. The embodiments of the present application image the dot matrix target ring light source on the dot matrix phase shift ring through the zone plate objective lens, and each point source image has a unique corresponding phase filter point on the dot matrix phase shift ring. Since the point source image and the phase filter point are relatively small, after the sample is placed, most of the low-order diffraction light can pass through the phase filter point without obstruction, thereby maximally eliminating the cause of the halo artifact and achieving the best effect of eliminating the halo artifact.

[0031] Furthermore, the embodiment of the present application only needs to take two sample images to conveniently and quickly perform quantitative imaging of the sample phase shift, and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Other features, objects and advantages of the present application will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0033] Figure 1 A schematic diagram of the structure of a phase contrast microscope provided in an embodiment of the present application;

[0034] Figure 2 A schematic diagram of the structure of a phase-shift quantitative imaging system provided in an embodiment of the present application.

[0035] Reference numerals:

[0036] 100-phase contrast microscope, 101-lattice target ring light source, 102-aperture diaphragm, 103-sample stage, 104-zone plate objective lens, 105-lattice phase shift ring, 106-detector;

[0037] 200 - phase-shift quantitative imaging system, 201 - phase-shift quantitative imaging device, 2011 - adjustment module, 2012 - shooting module, 2013 - calculation module. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0039] The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of this application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the application described can be implemented in an order other than those illustrated or described herein.

[0040] In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus that includes a series of steps or modules is not necessarily limited to those steps or modules explicitly listed, but may include other steps or modules not explicitly listed or inherent to these processes, methods, products, or apparatuses.

[0041] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0042] For ease of understanding and explanation, the following Figure 1 and Figure 2 The phase contrast microscope, phase shift quantitative imaging method, system and storage medium provided in the embodiments of the present application are described in detail.

[0043] Please refer to Figure 1 , which is a schematic diagram of the structure of a phase contrast microscope provided in an embodiment of the present application. The phase contrast microscope 100 comprises a dot matrix target ring light source 101, an aperture diaphragm 102, a sample stage 103, a zone plate objective lens 104, a dot matrix phase shift ring 105 and a detector 106 which are arranged in sequence.

[0044] It should be noted that, in the embodiment of the present application, the dot matrix target ring light source 101 is imaged on the dot matrix phase shift ring 105 through the zone plate objective lens 104, and each point source image has a unique corresponding phase filter point on the dot matrix phase shift ring 105, and each point source, the zone plate objective lens and the corresponding phase filter point can independently constitute a phase contrast microscope imaging optical path. In other words, there are as many independent and parallel phase contrast microscope imaging optical paths as there are point sources. Optionally, the dot matrix target ring light source 101 in the embodiment of the present application includes an X-ray source, and of course, it can also include light sources of other bands. Since the point source image and the phase filter point are relatively small, after the sample is placed, most of the low-order diffraction light can pass through the phase filter point without obstruction, thereby maximally eliminating the cause of the halo artifact and achieving the best effect of eliminating the halo artifact.

[0045] Assuming that the number of pixels of the detector 106 in the horizontal direction X and the vertical direction Y is N, then the width of the object plane field of view at the sample stage is also N pixels. According to the uncertainty relationship,

[0046]

[0047] In formula (1), δ represents the pixel diameter of the object plane where the sample is located, D represents the diameter of the zone plate objective lens, and d o represents the distance between the object plane at the sample stage and the zone plate objective, d i represents the distance between the detector and the zone plate objective, represents the angular width of the Fourier transform, then the minimum angular width is Furthermore, the angle between the first-order diffracted light and the zero-order transmitted light is If the diameter of the point source on the dot matrix target ring light source is smaller than The diameter of the phase shift filter point on the dot matrix phase shift ring is less than When S o S represents the distance between the dot matrix target ring light source and the zone plate objective lens. i Indicates the distance between the dot matrix phase shift ring and the zone plate objective lens.

[0048] The phase shift quantitative imaging method provided in the embodiment of the present application is described in detail below. The phase contrast microscope theory originates from the optical microscope, and according to the optical phase contrast microscope theory, before the phase shift ring is inserted, the phase contrast microscope is essentially an absorption contrast microscope, and the optical complex amplitude at the object plane at the sample stage 103 can be expressed as:

[0049]

[0050] Wherein, M represents the sample absorption, and Φ represents the phase shift of the sample. The optical complex amplitude at the image plane of the detector 106 can be expressed as:

[0051]

[0052]

[0053] Except for the different coordinates, equation (2) and equation (3) are completely the same. There is a similar magnification relationship between the image plane coordinates (X, Y) and the object plane coordinates (x, y). The imaging light intensity recorded by the detector 106 can be expressed as:

[0054]

[0055] In formula (3), the constant term represents the zero-order transmitted light of the sample. The complex amplitude of the imaging light after the phase shift of the zero-order transmitted light by the dot matrix phase shift ring can be expressed as:

[0056]

[0057] In formula (5), γ represents the absorption attenuation of the lattice phase shift ring, γ<1; φ represents the phase shift of the lattice phase shift ring. According to formula (5), the imaging light intensity can be obtained as:

[0058]

[0059] Equation (6) shows that after the insertion of the lattice phase shift ring, the phase difference between the zero-order transmitted light and the sample diffracted light is changed, so that the sample phase shift can modulate the imaging light intensity. When , formula (6) can be simplified as:

[0060]

[0061] Since general biological tissue samples are weakly absorbing and weakly phase shifting objects, equation (7) can be further simplified as:

[0062]

[0063] It should be noted that the mathematical derivation from equation (2) to equation (6) can be used for both optical phase contrast microscopy and X-ray phase contrast microscopy. Taking the dot matrix target ring light source 101 as an X-ray source as an example, since the refractive index is less than 1 in the X-ray band, the phase shift is a negative value, so The lattice phase shift ring is formed, and the last term of equation (7) is changed from positive to negative, that is:

[0064]

[0065] Further, simplifying formula (9) yields

[0066]

[0067] In formula (10), all the items on the right side of the equal sign are known quantities, and only Φ on the left side is an unknown quantity. Further, let U represent the known quantity on the right side of the equal sign of formula (10), and let γ = cotα, then formula (10) can be simplified to:

[0068] cosΦ+cotαsinΦ=U (11)

[0069] And transformed into:

[0070] sin(α+Φ)=Usinα (12)

[0071] By solving equation (12), the quantitative expression of phase shift Φ can be obtained as:

[0072]

[0073] In formula (13), γ = e -μt , μ and t represent the linear attenuation coefficient and thickness of the lattice phase shift ring, respectively. According to formula (13), the phase shift quantitative imaging method provided in the embodiment of the present application only needs to take two sample images, namely, one is the first sample image described by formula (9) Another second sample image I(X, Y) described by equation (4) can be used to obtain a quantitative image of the sample phase shift quickly and easily.

[0074] Furthermore, the specific steps of quantitative phase shift imaging of samples in the embodiment of the present application include: first, adjusting the imaging optical path of the phase contrast microscope 100 so that the center of the dot matrix target ring light source 101 and the center of the dot matrix phase shift ring 105 are located on the same optical axis, and the dot matrix target ring light source 101 is imaged on the dot matrix phase shift ring 105 through the zone plate objective 104, and each point source image is aligned with the corresponding phase filter point. Secondly, the first sample image when the dot matrix phase shift ring 105 is in place and the second sample image when the dot matrix phase shift ring 105 is removed are respectively photographed, the first sample image corresponds to equation (9), and the second sample image corresponds to equation (4). Finally, the sample phase shift image is calculated based on the first sample image and the second sample image, see equation (13).

[0075] Based on the above embodiments, the present application provides a phase shift quantitative imaging system. Figure 2 The phase-shift quantitative imaging system 200 includes Figure 1 The phase contrast microscope 100 and the phase shift quantitative imaging device 201 in the corresponding embodiment, wherein the phase shift quantitative imaging device 201 comprises:

[0076] The adjustment module 2011 is used to adjust the imaging optical path of the phase contrast microscope 100 so that the center of the dot matrix target ring light source 101 and the center of the dot matrix phase shift ring 105 are located on the same optical axis, and the dot matrix target ring light source 101 is imaged on the dot matrix phase shift ring 105 through the zone plate objective lens 104, and each point source image is aligned with the corresponding phase filter point;

[0077] A photographing module 2012 is used to photograph a first sample image when the dot matrix phase shift ring 105 is in place and a second sample image when the dot matrix phase shift ring 105 is removed;

[0078] The calculation module 2013 is used to calculate the sample phase-shift image according to the first sample image and the second sample image.

[0079] As another aspect, an embodiment of the present application provides a computer-readable storage medium for storing program code, wherein the program code is used to execute any one of the phase-shift quantitative imaging methods of the aforementioned embodiments.

[0080] It should be noted that, for the description of the same steps and the same contents in this embodiment as those in other embodiments, reference can be made to the description in other embodiments and will not be repeated here.

[0081] The embodiments of the present application provide a phase contrast microscope, a phase shift quantitative imaging method, a system and a storage medium, wherein the microscope includes a dot matrix target ring light source, an aperture, a sample stage, a zone plate objective lens, a dot matrix phase shift ring and a detector arranged in sequence. The embodiments of the present application image the dot matrix target ring light source on the dot matrix phase shift ring through the zone plate objective lens, and each point source image has a unique corresponding phase filter point on the dot matrix phase shift ring. Since the point source image and the phase filter point are relatively small, after the sample is placed, most of the low-order diffraction light can pass through the side of the phase filter point without obstruction, thereby maximally eliminating the cause of the halo artifact and achieving the best effect of eliminating the halo artifact. In addition, the embodiments of the present application only need to take two sample images to conveniently and quickly perform quantitative imaging of the sample phase shift, and the operation is simple.

[0082] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and modules described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0083] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the modules is only a logical function division. There may be other division methods in actual implementation, such as multiple modules or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or modules, which can be electrical, mechanical or other forms. The modules described as separate components may or may not be physically separated, and the components displayed as modules may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the modules can be selected according to actual needs to achieve the purpose of the scheme of this embodiment.

[0084] In addition, each functional module in each embodiment of the present application may be integrated into a processing unit, or each module may exist physically separately, or two or more units may be integrated into one module. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of a software functional unit. If the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it may be stored in a computer-readable storage medium.

[0085] Based on this understanding, the technical solution of the present application, or the part that contributes to the prior art, or all or part of the technical solution can be embodied in the form of a software product, which is stored in a storage medium and includes several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the phase-shift quantitative imaging method of each embodiment of the present application. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM), random access memory (RAM), disk or optical disk, etc., various media that can store program codes.

[0086] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A phase contrast microscope, characterized in that, the microscope comprises a dot matrix target ring light source, a pupil aperture, a sample stage, a zone plate objective lens, a dot matrix phase shift ring and a detector arranged in sequence, The diameter of the phase filtering point on the dot matrix phase shift ring is less than , Among them, represents the distance between the dot matrix phase shift ring and the zone plate objective lens, represents the diameter of the zone plate objective lens, represents the number of pixels corresponding to the field of view width of the object surface at the sample stage, represents the distance between the object surface and the zone plate objective lens at the sample stage; The diameter of the point source on the dot matrix target ring light source is less than , Among them, represents the distance between the dot matrix target ring light source and the zone plate objective lens.

2. The phase contrast microscope according to claim 1, characterized in that, the dot matrix target ring light source includes an X-ray source.

3. The phase contrast microscope according to claim 2, characterized in that, The phase shift of the dot matrix phase shift ring is .

4. A phase shift quantitative imaging method, characterized in that, the method is applied to the phase contrast microscope according to any one of claims 1 to 3, and includes: Adjusting the imaging optical path of the phase contrast microscope so that the center of the dot matrix target ring light source and the center of the dot matrix phase shift ring are on the same optical axis, and the dot matrix target ring light source is imaged on the dot matrix phase shift ring through the zone plate objective lens, and each point source image is aligned with the corresponding phase filtering point; Respectively taking a first sample image when the dot matrix phase shift ring is in place and a second sample image when the dot matrix phase shift ring is removed; Calculating a sample phase shift image according to the first sample image and the second sample image.

5. The phase shift quantitative imaging method according to claim 4, characterized in that, the imaging light intensity of the first sample image is: Among them, represents the absorption attenuation of the dot matrix phase shift ring, represents the phase shift of the sample.

6. The phase shift quantitative imaging method according to claim 5, characterized in that, the imaging light intensity of the second sample image is: ， where M represents sample absorption.

7. The phase shift quantitative imaging method according to claim 6, characterized in that, the sample phase shift corresponding to the sample phase shift image is calculated by the following formula: Among them, , and respectively represent the linear attenuation coefficient and the thickness of the dot matrix phase-shifting ring.

8. A phase shift quantitative imaging system, characterized in that, the system includes a phase shift quantitative imaging device and the phase contrast microscope according to any one of claims 1 to 3, and the phase shift quantitative imaging device includes: An adjustment module for adjusting the imaging optical path of the phase contrast microscope so that the center of the dot matrix target ring light source and the center of the dot matrix phase shift ring are on the same optical axis, and the dot matrix target ring light source is imaged on the dot matrix phase shift ring through the zone plate objective lens, and each point source image is aligned with the corresponding phase filtering point; A photographing module for respectively taking a first sample image when the dot matrix phase shift ring is in place and a second sample image when the dot matrix phase shift ring is removed; A calculation module for calculating a sample phase shift image according to the first sample image and the second sample image.

9. A computer-readable storage medium, characterized in that, the computer-readable storage medium stores one or more programs, and the one or more programs can be executed by one or more processors to implement the steps of the phase shift quantitative imaging method according to any one of claims 4 to 7.

Images