An interference phase imaging system and method and a three-dimensional shape reconstruction method

Abstract

The present invention discloses an interference phase imaging system, method and three-dimensional morphology reconstruction method, which include a laser, a first beam splitting prism, a second beam splitting prism, a first lens, a second lens, a third lens, a Wollaston prism, a sample, an aperture stop, an objective lens, a polarizer and a CCD; by using a beam splitting prism to split the laser beam, the present invention has a simple structure and is easy to operate; by adjusting the angle of the total reflection mirror to change the included angle between the two object beams incident on the sample, phase diagrams of incident light at different angles can be obtained, which is flexible and has strong applicability; through a symmetric optical path design, the two object beams have symmetry in the propagation path, ensuring the stability of the system of the present invention; two interference diagrams of the same sample in different incident directions can be obtained by one CCD, which has a simple structure, is beneficial to miniaturization of the device and cost reduction. The three-dimensional morphology reconstruction method disclosed by the present invention requires a small amount of data, is simple to calculate, has a fast speed, and is beneficial to real-time imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of quantitative phase imaging, and in particular relates to an interference phase imaging system and method and a three-dimensional morphology reconstruction method. Background Art

[0002] In recent years, observing transparent biological samples using quantitative phase imaging (QPI) technology has the characteristics of label-free, fast, high resolution, etc., and is therefore widely used in biomedical and other fields. Interference phase imaging recovers the phase information of a sample from the interference intensity distribution, and the phase data carries the thickness and refractive index distribution information of the sample in the light propagation direction, which can reflect its morphological structure and internal components, and has great potential in medical diagnosis and biological research. However, in QPI, the thickness and refractive index of the sample are coupled, resulting in the morphological features not being directly displayed, and it is necessary to reconstruct its three-dimensional morphology. Most existing algorithms require collecting the phase information of the sample from multiple angles and going through cumbersome inversion calculations to obtain. And the current clinical testing technology is constantly developing in the direction of less sample volume, higher sensitivity, faster speed, and more automated analysis, and the demand for miniaturization of detection equipment and simplification of operation is getting higher and higher. There are mainly two ways to obtain two images of the same sample in different incident light directions: one is the change of the sample's own attitude angle, and the other is to change the incident light direction. If the scheme of rotating the sample is adopted, the construction of the imaging optical path itself will be relatively simple. Theoretically, the change of the angle relationship between the sample and the incident light can be achieved by loading an optical tweezer to control the position and attitude of the cell or rotating the sample slide as a whole, but the accuracy of sampling is difficult to guarantee, and rotating the slide as a whole will also increase some uncertain factors. Especially for suspended cells, such as white blood cells, when preparing the sample, the supernatant will be diluted, but it is difficult to ensure that there is only one cell in the field of view. The obtained phase map often contains multiple samples. When rotating the cell slide, multiple cells will flow in the culture solution. Not only the flipping angles of each cell itself cannot be controlled and determined, but also the relative positions between cells will change and rearrange in the field of view, which brings great difficulties to target positioning and corresponding sampling.

[0003] According to the different incident light angles, the phase imaging system can be divided into two categories: the orthogonal phase imaging system and the non-orthogonal phase imaging system. The relevant reconstruction algorithms designed based on the orthogonal phase imaging system can decouple the cell sample models with multiple different kernels. However, during the experiment, this method has relatively strict requirements for the optical path setup and debugging. If the single-channel imaging method is adopted, due to the limitation of the numerical aperture of the traditional lens, the maximum value of the light-receiving angle generally does not exceed 60°, and it is very difficult to split the incident light by 90°. If the dual-channel imaging method is adopted, the two object lights must be perpendicular to each other, and the arrangement methods of the light arms of the object light and the reference light and even the signal reception are greatly restricted, which is not conducive to the miniaturization of the instrument and equipment. Moreover, the dual-channel dual-CCD signal acquisition may introduce more complex noise and errors while increasing the cost. Summary of the Invention

[0004] Aiming at the above technical problems, one of the purposes of one embodiment of the present invention is to provide an interference phase imaging system and method. This system can be applicable to both orthogonal and non-orthogonal phase imaging optical paths at the same time, and a single CCD is used to collect the dual-channel optical information. The collected phase map can realize the fast phase imaging of the object after being processed. One of the purposes of one embodiment of the present invention is to provide a three-dimensional shape reconstruction method according to the interference phase imaging system. This three-dimensional shape reconstruction method adopts the shape reconstruction algorithm under non-orthogonal phase data.

[0005] Note that the recording of these purposes does not prevent the existence of other purposes. One embodiment of the present invention does not need to achieve all the above purposes. Purposes other than the above can be extracted from the descriptions of the specification, drawings, and claims.

[0006] The present invention adopts the idea of single CCD multiplexing to design an angle-adjustable dual-channel optical phase information acquisition system, and mainly focuses on non-orthogonal imaging. Incident light rays from different directions not only have different optical paths in the cells to be measured, but also the optical paths experienced in the glass slide and the environmental liquid will vary due to different incident paths. These differences will accumulate in the phase distribution data, thus affecting the standardization and normalization of the phase distribution reference plane. To minimize the error as much as possible, the present invention adopts a symmetric optical path design. And on this basis, a three-dimensional shape fast reconstruction method based on non-orthogonal phase data is proposed.

[0007] The present invention adopts an interference phase imaging system with dual-channel synchronous acquisition and a corresponding algorithm for rapidly reconstructing the three-dimensional morphology of a sample based on non-orthogonal phase data. Based on the Mach-Zehnder interference principle and the idea of polarization splitting, it realizes the synchronous acquisition of dual-channel optical information through a single CCD. The laser is divided into object light and reference light by a beam splitter. The object light is separated into two polarization components after passing through a Wollaston prism, and then respectively passes through total reflection mirrors and simultaneously transmits through the sample along symmetric paths. The reference light and the two object lights with different polarization components and phase shifts are combined by the beam splitter and propagated in a common optical path manner. The object and reference light beams with the same polarization component interfere with each other. By selecting different polarization components to pass through the polarizer, two sets of interference patterns are acquired by a CCD camera, and then two phase diagrams of the same sample in different incident directions are obtained through Hilbert transform respectively. Based on the two phase diagrams, the sample morphology is reconstructed. Edge detection is performed on the phase diagrams respectively to obtain the pixel coordinates of the boundary points of the sample structure on the corresponding projection planes. The direct linear transformation is used to select control points to establish the relationship between its pixel coordinates and the object space coordinates. The object space coordinates corresponding to the pixel coordinates of the point to be determined are obtained from the obtained relationship coefficients, and then the three-dimensional morphology model of the sample is reconstructed. The present invention adjusts the angle of the total reflection mirror to change the angles of the two object lights incident on the sample, so as to achieve orthogonal and non-orthogonal phase imaging. The present invention has wide practical value and application prospects in quantitative phase imaging, especially in the application field of biological cell morphology recognition.

[0008] The present invention adopts the idea of polarization splitting, constructs a symmetric common optical path design based on the Mach-Zehnder interference principle, and proposes an interference phase imaging system for dual-channel optical information acquisition.

[0009] The above technical object of the present invention is achieved by the following technical means.

[0010] An interference phase imaging system includes a laser, a first beam splitter prism, a second beam splitter prism, a first lens, a second lens, a third lens, a Wollaston prism, a sample, an aperture stop, an objective lens, a polarizer, and a CCD;

[0011] A first total reflection mirror and a second total reflection mirror are successively arranged on the optical path between the laser and the first beam splitting prism; a third total reflection mirror is arranged on the optical path between the first beam splitting prism and the Wollaston prism; symmetrically distributed fourth total reflection mirror and fifth total reflection mirror are arranged on the optical path between the Wollaston prism and the sample; a sixth total reflection mirror is arranged on the optical path between the first beam splitting prism and the first lens; along the output direction of the laser, the incident light successively passes through the first total reflection mirror and the second total reflection mirror and then is split into an object light O and a reference light R by the first beam splitting prism; the object light O is reflected by the third total reflection mirror to the Wollaston prism to separate the two polarization components, and the two separated components respectively pass through the fourth total reflection mirror and the fifth total reflection mirror and are transmitted out of the sample along a symmetric path, enter the objective lens, and after passing through the objective lens, two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R that has successively passed through the sixth total reflection mirror and the first lens in the second beam splitting prism. The two polarization components in the object light 0 respectively interfere with the two polarization components in the reference light R to obtain two different sets of interference fringes. Different polarization components are selected to pass through by the second lens, the third lens and the polarizer, and two interference patterns are obtained on a CCD.

[0012] In the above solution, an aperture stop is further included; the aperture stop is located between the sample and the objective lens.

[0013] An imaging method of an interference phase imaging system according to the dual-channel optical information acquisition includes the following steps:

[0014] The laser beam passes through the first total reflection mirror and the second total reflection mirror along the output direction of the laser and then enters the first beam splitting prism and is split into an object light O and a reference light R. After the object light O is reflected by the third total reflection mirror to the Wollaston prism, it is separated into two polarization components, namely a parallel component and a perpendicular component, at a specific polarization splitting angle. The two separated polarization components respectively pass through the fourth total reflection mirror and the fifth total reflection mirror and are simultaneously transmitted out of the sample along a symmetric path and enter the objective lens;

[0015] Two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R in the second beam splitting prism. The parallel component and the perpendicular component of the object light 0 after polarization respectively interfere with the corresponding two polarization components in the reference light R to obtain two different sets of interference fringes. The combined beam successively passes through the second lens, the third lens and the polarizer. By selecting different polarization components, different polarization components pass through, and two interference patterns of the same sample in different incident directions are collected by a CCD.

[0016] In the above solution, the structure of the Wollaston prism is adjusted to separate the two polarization components in the object light 0 at a specific angle, and the two separated object lights have symmetry in the propagation path.

[0017] In the above solution, the fourth total reflection mirror and the fifth total reflection mirror are two total reflection mirrors with adjustable angles. By adjusting the angles of the fourth total reflection mirror and the fifth total reflection mirror, the included angle between the two object lights incident on the sample is changed, so as to realize the incident of the two incident lights in the orthogonal direction and the non-orthogonal direction.

[0018] In the above solution, the two separated components respectively pass through the fourth total reflection mirror and the fifth total reflection mirror and are transmitted out of the sample along the symmetric path, and enter the objective lens through the aperture stop.

[0019] In the above solution, the two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R through the second beam splitting prism. The two polarization components in the object light 0 respectively interfere with the two polarization components in the reference light R to obtain two different sets of interference fringes.

[0020] In the above solution, the second lens and the third lens form an optical 4F system.

[0021] A three-dimensional shape reconstruction method for an interference phase imaging system according to the above-described dual-path optical information acquisition includes the following steps:

[0022] Step S1: Adjust the fourth total reflection mirror and the fifth total reflection mirror of the interference phase imaging system for dual-path optical information acquisition, so that the included angle α between the two incident lights and the sample is not equal to 90°, and two phase diagrams are obtained on the CCD;

[0023] Step S2: Input the two phase diagrams obtained in step S1 into a computer, perform edge detection on the two phase diagrams respectively, extract the pixel coordinates of the boundary points, and establish a linear relationship between the two-dimensional plane pixel coordinates and their corresponding object space coordinates through direct linear transformation:

[0024]

[0025] In the formula, x and y represent pixel coordinates, X, Y and Z represent object space coordinates, and l i represents the relationship coefficient between the pixel coordinates and the object space coordinates, where i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11;

[0026] Step S3: Select N control points from the pixel coordinates of the edge points extracted after the edge detection of the two images respectively according to the principle of uniform spatial dispersion. Taking the coordinates of the pixel points as the observed values, and assuming the correction value of the observed value of the control point pixel coordinates is (v x , v y) If the correction values of the systematic error are (Δx, Δy), then we have:

[0027]

[0028] In the formula, k1 and k2 are symmetric radial distortion coefficients, r is the radial vector of the image point. Let A = l9X + l 10 Y + l 11 Z + 1. Taking l i and k1 as unknowns, list the error equations:

[0029]

[0030] Denoted as V = Ml i - W, then the relationship coefficient l i between the pixel coordinates and the object space coordinates is obtained as l T = (M -1 M) T M

[0031] Step S4: Assume the unknown object space coordinates as the points to be determined. Let the correction of the observed value of the pixel coordinates of the points to be determined be Then:

[0032]

[0033] Let A = l9X + l 10 Y + l 11 Z + 1, then:

[0034]

[0035] Denoted as V = NS - Q, then S = (N T N) -1 N T Q, is the average value of the pixel coordinates, and represent the correction of the observed value of the pixel coordinates, l i represents the relationship coefficient between the pixel coordinates and the object space coordinates, i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. That is, the object space coordinates corresponding to the pixel coordinates of the points to be determined are obtained, and thus the three-dimensional shape of the sample is obtained.

[0036] In the above solution, in step S2, Matlab software is used for three-dimensional shape reconstruction.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] According to one aspect of the present invention, by using a beam splitting prism to split a laser beam, the structure is simple and easy to operate; by adjusting the angle of the total reflection mirror to change the included angle between the two object beams incident on the sample, phase diagrams of incident light at different angles can be obtained, which is flexible and highly applicable; through a symmetric optical path design, the two object beams have symmetry in the propagation path, ensuring the stability of the system of the present invention; by using one CCD, two interference diagrams of the same sample in different incident directions can be obtained, with a simple structure, which is beneficial to miniaturization of the device and cost reduction. According to one aspect of the present invention, the morphological reconstruction algorithm of the three-dimensional morphological reconstruction method requires a small amount of data, is computationally simple, fast, and is beneficial to real-time imaging.

[0039] Note that the description of these effects does not preclude the existence of other effects. One aspect of the present invention does not necessarily have to have all of the above effects. Effects other than the above can be obviously seen and extracted from the descriptions in the specification, drawings, claims, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a schematic optical path diagram of an interference phase imaging system according to an embodiment of the present invention.

[0041] Figure 2 is a schematic diagram of a double-medium concentric sphere model according to an embodiment.

[0042] Figure 3 is a phase edge detection diagram of a double-medium concentric sphere model according to an embodiment.

[0043] Figure 4 is a reconstructed three-dimensional morphology diagram of a double-medium concentric sphere model according to an embodiment.

[0044] Figure 5 is an experimental phase diagram of a polystyrene microsphere according to an embodiment, where Figure 5 (a) is the phase diagram in the X direction, Figure 5 (b) is the phase diagram at an angle of 60° with the X direction.

[0045] Figure 6 is a reconstructed three-dimensional morphology diagram of a polystyrene microsphere according to an embodiment.

[0046] In the figure: 1. Laser; 2. First total reflection mirror; 3. Second total reflection mirror; 4. Third total reflection mirror; 5. Sixth total reflection mirror; 6. Fourth total reflection mirror; 7. Fifth total reflection mirror; 8. First beam splitting prism; 9. Second beam splitting prism; 10. First lens; 11. Second lens; 12. Third lens; 13. Wollaston prism; 14. Sample; 15. Aperture stop; 16. Objective lens; 17. Polarizer; 18. CCD. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, where like or similar reference numerals designate like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention.

[0048] In the description of the present invention, it should be understood that the orientation or positional relationships indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "front", "rear", "left", "right", "upper", "lower", "axial", "radial", "vertical", "horizontal", "inner", "outer", etc. are based on the orientation or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, "a plurality of" means two or more unless otherwise specifically defined.

[0049] In the present invention, unless otherwise clearly defined and limited, the terms "mounted", "connected", "coupled", "fixed", etc. should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0050] Figure 1 Shown is a preferred embodiment of the interference phase imaging system. The interference phase imaging system is an interference phase imaging system for dual-channel optical information acquisition, and includes a laser 1, a first beam splitting prism 8, a second beam splitting prism 9, a first lens 10, a second lens 11, a third lens 12, a Wollaston prism 13, a sample 14, an aperture stop 15, an objective lens 16, a polarizer 17, and a CCD 18.

[0051] A first total reflection mirror 2 and a second total reflection mirror 3 are successively provided on the optical path between the laser 1 and the first beam splitting prism 8; a third total reflection mirror 4 is provided on the optical path between the first beam splitting prism 8 and the Wollaston prism 13; symmetrically distributed fourth total reflection mirror 6 and fifth total reflection mirror 7 are provided on the optical path between the Wollaston prism 13 and the sample 14; a sixth total reflection mirror 5 is provided on the optical path between the first beam splitting prism 8 and the first lens 10; along the output direction of the laser 1, the incident light successively passes through the first total reflection mirror 2 and the second total reflection mirror 3 and then is split by the first beam splitting prism 8 into object light O and reference light R; the object light O is reflected by the third total reflection mirror 4 to the Wollaston prism 13 to separate the two polarization components, and the two separated components respectively pass through the fourth total reflection mirror 6 and the fifth total reflection mirror 7 and transmit out of the sample 14 along a symmetric path, and after passing through the aperture stop 15 to reduce the astigmatism caused by oblique incidence, enter the objective lens 16. After passing through the objective lens 16, the two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R that successively passes through the sixth total reflection mirror 5 and the first lens 10 in the second beam splitting prism 9. The two polarization components in the object light 0 respectively interfere with the two polarization components in the reference light R to obtain two different sets of interference fringes. By selecting different polarization components through the second lens 11, the third lens 12 and the polarizer 17, two interference patterns of the same sample 14 in different incident directions are obtained on a CCD 18.

[0052] An imaging method of an interference phase imaging system according to the dual-channel optical information acquisition includes the following steps:

[0053] The laser beam passes through the first total reflection mirror 2 and the second total reflection mirror 3 along the output direction of the laser 1 and then enters the first beam splitting prism 8 and is split into object light O and reference light R. After the object light O is reflected by the third total reflection mirror 4 to the Wollaston prism 13, it is separated into two polarization components, namely the parallel component and the perpendicular component, according to a specific polarization splitting angle. The two separated polarization components respectively pass through the fourth total reflection mirror 6 and the fifth total reflection mirror 7 and simultaneously transmit out of the sample 14 along a symmetric path. To reduce the astigmatism caused by oblique incidence, an aperture stop 15 is placed in front of the objective lens 16. After the two object lights transmit out of the sample 14, they enter the objective lens 16 through the aperture stop 15;

[0054] The two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R in the second beam splitting prism 9. The parallel component and the perpendicular component of the object light 0 after polarization respectively interfere with the corresponding two polarization components in the reference light R to obtain two different sets of interference fringes. The combined beam successively passes through the second lens 11, the third lens 12 and the polarizer 17. By selecting different polarization components, different polarization components pass through, and two interference patterns of the same sample 14 in different incident directions are collected by a CCD 18.

[0055] Adjust the structure of the Wollaston prism 13 so that the two polarization components in the object light 0 are separated by a specific angle, and the two separated object lights are symmetric in the propagation path, which can avoid errors caused by different optical paths of the two object lights.

[0056] The fourth total reflection mirror 6 and the fifth total reflection mirror 7 are two total reflection mirrors with adjustable angles. By adjusting the angles of the fourth total reflection mirror 6 and the fifth total reflection mirror 7, the included angle between the two object lights incident on the sample 14 can be changed, and orthogonal incidence and non-orthogonal incidence of the two incident lights can be achieved.

[0057] The aperture stop 15 is used to reduce the astigmatism caused by oblique incidence, so that the final imaging range is within the CCD recording target surface.

[0058] The two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R through the second beam splitter prism 9. The two polarization components in the object light interfere with the two polarization components in the reference light R respectively, and two different interference fringes can be obtained.

[0059] The second lens 11 and the third lens 12 form an optical 4F system, and a polarizer 17 is added after it, which can realize the extraction of information of different polarization components.

[0060] The imaging device only uses one CCD 18, which can avoid errors caused by different sampling devices and reduce the sampling cost.

[0061] According to the three-dimensional shape reconstruction method of the interference phase imaging system for dual-path optical information acquisition, the method includes the following steps:

[0062] Step S1: Adjust the fourth total reflection mirror 6 and the fifth total reflection mirror 7 of the interference phase imaging system for dual-path optical information acquisition, so that the included angle α between the two incident lights and the sample 14 is not equal to 90°, and two phase diagrams are obtained on the CCD 18.

[0063] Step S2: Input the two phase diagrams obtained in step S1 into a computer, perform edge detection on the two phase diagrams respectively through the shape reconstruction module of matlab, extract the pixel coordinates of the boundary points, and establish a linear relationship between the two-dimensional plane pixel coordinates and their corresponding object space coordinates through direct linear transformation:

[0064]

[0065] In the formula, x and y represent pixel coordinates, X, Y and Z represent object space coordinates, l iCoefficients representing the relationship between pixel coordinates and object space coordinates, where i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11;

[0066] Step S3: Select N control points from the edge point pixel coordinates extracted after edge detection of the two images based on the principle of uniform spatial dispersion. Taking the coordinates of the pixel points as observed values, let the correction values of the observed values of the control point pixel coordinates be (v x , v y ), and the correction values of the systematic errors be (Δx, Δy). Then we have:

[0067]

[0068] In the formula, k1 and k2 are symmetric radial distortion coefficients, r is the radius vector of the image point. Let A = l9X + l 10 Y + l 11 Z + 1. Taking l i and k1 as unknowns, list the error equations:

[0069]

[0070] Denoted as V = Ml i - W, then the relationship coefficients l i = (M T M) -1 M T W, i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11;

[0071] Step S4: Let the unknown object space coordinates be the points to be determined. Let the correction of the observed value of the pixel coordinates of the points to be determined be Then:

[0072]

[0073] Let A = l9X + l 10 Y + l 11 Z + 1, then:

[0074]

[0075] Denoted as V = NS - Q, then S = (N T N) -1 N T Q, is the average value of the pixel coordinates, and represent the corrections of the observed values of the pixel coordinates, l iDenote the relationship coefficients between pixel coordinates and object space coordinates, where i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. That is, the object space coordinates corresponding to the pixel coordinates of the point to be determined are obtained, thereby obtaining the three-dimensional shape of sample 14.

[0076] In step S2, Matlab software is used for three-dimensional shape reconstruction. Specific embodiment 1:

[0078] The three-dimensional shape reconstruction method of the present invention is a reconstruction algorithm for an interference phase imaging system with dual-path optical information acquisition under non-orthogonal phase data. Taking the dual-medium concentric sphere model as an example, it is described as follows:

[0079] The angles ∝ between the directions of the two incident light beams and sample 14 are 60°. The geometric size of the diffraction surface is 16 μm × 16 μm, the pixel size is 256 × 256, the radius of the cytoplasm is 4 μm, the radius of the cell nucleus is 2 μm, the cytoplasm and the cell nucleus are concentric, and the center coordinates of the sphere are (0, 0, 0). Its three-dimensional model is as Figure 2 shown. The wavelength of the incident light is 632.8 nm. Let the refractive index of the environmental liquid be 1.339, the refractive index of the cytoplasm be 1.360, and the refractive index of the cell nucleus be 1.450. Two phase diagrams obtained by irradiating from the X-axis direction and the direction at an angle of 60° with the X-axis. Since the model is a dual-medium concentric sphere, the two phase diagrams obtained by irradiating from these two directions are the same. The Canny operator is used to detect the edges of the two phase diagrams respectively, and an edge distribution diagram is obtained, as Figure 3 shown. According to the edge detection results, boundary points are obtained. 156 control points are selected on the principle of uniform spatial dispersion. The coordinates of some control points are shown in Table 1. The relationship coefficients l between pixel coordinates and object space coordinates can be calculated by formula (1) i .

[0080] Table 1 Coordinates of some control points

[0081]

[0082]

[0083] In the formula, (x, y) represents pixel coordinates, (x0, y0) represents the coordinates of the principal point of the image, (X, Y, Z) represents object space coordinates, and l i The coefficient represents the relationship coefficient between pixel coordinates and object space coordinates, where i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11.

[0084] For the approximate solution of object space coordinates, the object space coordinates are used as unknowns. From the known pixel coordinates of the point to be determined, the approximate values of the corresponding object space coordinates can be calculated by formula (2). The pixel coordinates of the point to be determined are shown in Table 2.

[0085]

[0086] Solve the exact value of the object space coordinates of the to-be-determined point again, using the pixel coordinates of the to-be-determined point as the observed values. Let the correction number of the observed value of the pixel coordinates of the to-be-determined point be There is:

[0087]

[0088] The exact value of the object space coordinates corresponding to the pixel coordinates of the to-be-determined point can be obtained. The three-dimensional shape of the reconstructed double-medium concentric sphere model is as Figure 4 shown. It can be seen from Figure 4 that the diameter of the reconstructed phase cell cytoplasm is 7.9703 μm, with an error of 0.37%; the diameter of the cell nucleus is 4.048, with an error of 1.2%. The results show that this method can be applied to the measurement and calculation of micron-scale objects.

[0089] Table 2 Pixel coordinates of the to-be-determined point

[0090] Specific embodiment 2:

[0092] Select polystyrene microspheres as samples through experiments. The included angle ∝ between the directions of two incident light beams and the sample 14 is 60°. The environmental liquid is Cargille refractive index matching liquid. The refractive index of the sample is 1.5916, the refractive index of the environmental liquid is 1.4218, and the wavelength of the incident light is 632.8 nm. The obtained experimental phase diagram is as Figure 5 shown, where Figure 5 (a) is the phase diagram in the X direction, Figure 5 (b) is the phase diagram at an angle of 60° with the X direction.

[0093] Table 3 Error analysis

[0094]

[0095] The polystyrene microspheres reconstructed by the reconstruction algorithm of the present invention are as Figure 6 shown, and by Figure 6 and Figure 5 After error analysis, the results are shown in Table 3. The diameter measurement errors along the Z direction and the Y direction are 0.64% and 1.63% respectively. The results show that only through Figure 5 a and Figure 5 b two phase diagrams, the present invention can preferably reconstruct the three-dimensional shape of the sample as Figure 6 shown.

[0096] It should be understood that although this specification is described according to various embodiments, not every embodiment contains only one independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0097] The series of detailed descriptions listed above are only specific descriptions of the feasible embodiments of the present invention, and they are not intended to limit the protection scope of the present invention. Any equivalent embodiments or changes made without departing from the spirit of the present invention should be included within the protection scope of the present invention.

Claims

1. An interference phase imaging system, characterized in that, It includes a laser (1), a first beam-splitting prism (8), a second beam-splitting prism (9), a first lens (10), a second lens (11), a third lens (12), a Wollaston prism (13), a sample (14), an aperture stop (15), an objective lens (16), a polarizer (17) and a CCD (18); A first total reflection mirror (2) and a second total reflection mirror (3) are sequentially arranged on the optical path between the laser (1) and the first beam-splitting prism (8); a third total reflection mirror (4) is arranged on the optical path between the first beam-splitting prism (8) and the Wollaston prism (13); a symmetrically distributed fourth total reflection mirror (6) and a fifth total reflection mirror (7) are arranged on the optical path between the Wollaston prism (13) and the sample (14); a sixth total reflection mirror (5) is arranged on the optical path between the first beam-splitting prism (8) and the first lens (10); along the output direction of the laser (1), the incident light sequentially passes through the first total reflection mirror (2) and the second total reflection mirror (3) and then is split by the first beam-splitting prism (8) into an object light O and a reference light R; the object light O is reflected by the third total reflection mirror (4) to the Wollaston prism (13), and the two polarization components are separated. The two separated components then respectively pass through the fourth total reflection mirror (6) and the fifth total reflection mirror (7) and transmit out of the sample (14) along a symmetric path, enter the objective lens (16). After passing through the objective lens (16), two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet with the reference light R that sequentially passes through the sixth total reflection mirror (5) and the first lens (10). The two polarization components in the object light 0 respectively interfere with the two polarization components in the reference light R, obtaining two sets of different interference fringes. By selecting different polarization components to pass through the second lens (11), the third lens (12) and the polarizer (17), two interference patterns are obtained on a CCD (18).

2. The interference phase imaging system according to claim 1, wherein It further includes an aperture stop (15); the aperture stop (15) is located between the sample (14) and the objective lens (16).

3. An imaging method for the interference phase imaging system according to claim 1 or 2, characterized in that, It includes the following steps: The laser beam passes through the first total reflection mirror (2) and the second total reflection mirror (3) along the output direction of the laser (1) and then enters the first beam-splitting prism (8) and is split into an object light O and a reference light R. After the object light O is reflected by the third total reflection mirror (4) to the Wollaston prism (13), it is separated into two polarization components, namely a parallel component and a perpendicular component, at a specific polarization splitting angle. The two separated polarization components respectively pass through the fourth total reflection mirror (6) and the fifth total reflection mirror (7) and simultaneously transmit out of the sample (14) along a symmetric path and enter the objective lens (16); Two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R through the second beam splitter prism (9). The parallel component and the perpendicular component of the object light 0 after polarization interfere with the corresponding two polarization components in the reference light R respectively to obtain two different sets of interference fringes. The combined beam of light passes through the second lens (11), the third lens (12) and the polarizer (17) in sequence. By selecting different polarization components, different polarization components pass through, and two interference patterns of the same sample in different incident directions are collected by a CCD (18).

4. The imaging method of the interference phase imaging system according to claim 3, wherein the structure of the Wollaston prism (13) is adjusted to separate the two polarization components in the object light 0 at a specific angle, and the two separated object lights are symmetric in the propagation path.

5. The imaging method of the interference phase imaging system according to claim 3, wherein the fourth total reflection mirror (6) and the fifth total reflection mirror (7) are two total reflection mirrors with adjustable angles. By adjusting the angles of the fourth total reflection mirror (6) and the fifth total reflection mirror (7), the included angle between the two object lights incident on the sample (14) is changed to achieve the incident of the two incident lights in the orthogonal direction and the non-orthogonal direction.

6. The imaging method of the interference phase imaging system according to claim 3, characterized in that, The two separated components respectively pass through the fourth total reflection mirror (6) and the fifth total reflection mirror (7) and transmit out of the sample (14) along the symmetric path, and enter the objective lens (16) through the aperture stop (15).

7. The imaging method of the interference phase imaging system according to claim 3, characterized in that, The two linearly polarized object lights with the same direction, carrying different phase information and perpendicular vibration directions meet the reference light R through the second beam splitter prism (9). The two polarization components in the object light 0 interfere with the two polarization components in the reference light R respectively to obtain two different sets of interference fringes.

8. The imaging method of the interference phase imaging system according to claim 3, characterized in that, The second lens (11) and the third lens (12) form an optical 4F system.

9. The three-dimensional morphological reconstruction method of the interference phase imaging system according to claim 1 or 2, characterized in that including the following steps: Step S1: Adjust the fourth total reflection mirror (6) and the fifth total reflection mirror (7) of the interference phase imaging system for collecting dual-channel optical information, so that the included angle α between the two incident lights and the sample (14) is not equal to 90°, and two phase diagrams are obtained on the CCD (18); Step S2: Input the two phase diagrams obtained in Step S1 into a computer, perform edge detection on the two phase diagrams respectively, extract the pixel coordinates of the boundary points, and establish a linear relationship between the two-dimensional plane pixel coordinates and their corresponding object space coordinates through direct linear transformation: where x and y represent pixel coordinates, X, Y, and Z represent object space coordinates, and l i represents the relationship coefficient between the pixel coordinates and the object space coordinates, where i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11; Step S3: Select N control points from the pixel coordinates of the edge points extracted after edge detection for the two images. Taking the coordinates of the pixel points as the observed values, let the correction values of the observed values of the pixel coordinates of the control points be (v x , v y ), and the correction values of the systematic errors be (Δx, Δy). Then we have: wherein: In the formula, k1 and k2 are symmetric radial distortion coefficients, r is the radius vector of the image point, and (x0, y0) represents the coordinates of the image principal point. Let \(A = l9X + l\) 10 Y + l 11 Z + 1, with \(l\) i and \(k1\) as unknowns, list the error equations: Denote it as V = Ml i -W, then the relationship coefficient l between the pixel coordinates and the object space coordinates is obtained i =(M T M) -1 M T W, i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11; Step S4. Set the unknown object space coordinates as the to-be-determined points, and set the correction of the observed values of the pixel coordinates of the to-be-determined points as Then: Let A = l9X + l 10 Y + l 11 Z + 1, then: Denote V = NS - Q, then S = (N T N) -1 N T Q, which is the average value of pixel coordinates, and represents the correction of the observed value of pixel coordinates, l i represents the relationship coefficient between pixel coordinates and object space coordinates, i = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, that is, the object space coordinates corresponding to the pixel coordinates of the point to be determined are obtained, so as to obtain the three-dimensional shape of the sample (14).

10. The three-dimensional morphology reconstruction method of the interference phase imaging system according to claim 9, characterized in that, In Step S2, the matlab software is used for three-dimensional shape reconstruction.

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