A high-order non-interference quantitative phase imaging system and method based on single exposure

Abstract

The present invention discloses a single-exposure high-order non-interference quantitative phase imaging system and method, belonging to the field of optical microscopic imaging technology, including a microscope module and a spectroscopic modulation module arranged in sequence, the microscope module including a halogen lamp, a condenser, an annular aperture, a sample, an objective lens, a phase plate, a lens one, a reflector and a lens two arranged in sequence along the light beam propagation direction, and the spectroscopic modulation module including a lens three, a spectroscopic module one, a spectroscopic module two, a spatial light modulator, a lens four and a CMOS camera arranged in sequence along the light beam propagation direction. The present invention adopts the above-mentioned single-exposure high-order non-interference quantitative phase imaging system, retaining the advantages of the white light illumination system such as stable structure, insensitivity to environmental interference, high phase sensitivity and low spatial noise.

Description

Technical Field

[0001] The present invention relates to the field of optical microscopy technology, and in particular to a single-exposure high-order non-interference quantitative phase imaging system and method. Background Art

[0002] The absorption coefficient of the internal structure of biological cells is very small, close to colorless and transparent, making it difficult to observe the details inside the cells. This also leads to slight changes in optical thickness, which has almost no effect on light intensity. Therefore, the commonly used methods for observing the internal structure and physiological state of cells usually rely on staining, fluorescent labeling and high-intensity light excitation, but this will interfere with the natural functions of cells. Therefore, a label-free imaging technology that can reliably identify the internal structure of cells without affecting the normal physiological functions of cells is particularly important.

[0003] Quantitative phase imaging technology has become an important tool for studying transparent samples such as cells due to its non-contact, non-destructive, and label-free measurement method. However, due to the limitation of the illumination source, the initial quantitative phase imaging was largely limited to red blood cell imaging or the evaluation of overall cell parameters, and the strong speckle noise in the laser light source-based system seriously affected the imaging quality. In the 21st century, Professor G. Popescu combined Zernike's phase contrast microscopy technology with Gabor's holography and proposed the spatial light interference microscopy (SLIM) imaging technology. This method records the phase information by collecting the phase contrast image of the sample and then reproduces it through holography. The imaging technology system uses white light and annular light sources for illumination, eliminating the influence of speckle noise. However, for samples with phase values ​​exceeding 2π, spatial light interference microscopy requires phase unwrapping, and it is difficult to directly obtain the absolute distribution of the phase. Therefore, we can consider using a phase recovery algorithm that does not require unwrapping.

[0004] The light intensity transport equation method is a typical direct phase retrieval method. Light waves undergo a natural phase-to-intensity conversion during propagation, which can be described by the intensity transport equation. The light intensity transport differential equation (TIE) is a second-order elliptic partial differential equation that is related to the quantitative change in the phase of light waves propagating in a plane perpendicular to the optical axis. When the axial differential of the light intensity and the light intensity distribution are obtained, the phase information can be directly obtained by numerically solving the light intensity transport equation. This equation relates the amplitude and intensity of the light wave field, providing us with a method to infer the phase distribution from known intensity information. Compared with the interference method and iterative phase retrieval method, its main features include two points: (1) The absolute distribution of the phase can be obtained without phase unwrapping, so the 2π phase wrapping problem in interferometric measurement will not occur during the phase recovery process; (2) The phase recovery method is a non-iterative method, and the phase is obtained by directly solving the differential equation.

[0005] In 1988, Ichikawa et al. experimentally verified the validity of the intensity transfer equation for the first time. They used the fast Fourier transform (FFT) to solve the equation and successfully obtained the phase distribution of the light wave field. This work laid the foundation for subsequent research. In 1995, Gureyev et al. established the adaptability and uniqueness of the solution of the intensity transfer equation through rigorous mathematical proofs, further promoting research in this field. In 1996, they proposed a solution for orthogonal polynomial decomposition. In 1997, Nugent demonstrated the prior conditions of the FFT solution (uniformity of light intensity distribution), providing a deeper understanding of the applicability of the intensity transfer equation.

[0006] Entering the 21st century, researchers continued to improve the theory and application of TIE. For example, Allen et al. proposed a solution based on Green's function in 2000, and Sheppard performed Born approximation analysis on the equation in 2002. In 2010, Waller et al. proposed a high-order intensity transfer equation by collecting intensity information of multiple defocused planes, which improved the accuracy and anti-noise performance of phase recovery. In the same year, Kou et al. combined the intensity transfer equation with differential interference contrast technology to achieve quantitative phase recovery. In 2012, Zuo et al. derived a generalized intensity transfer equation under partially coherent light fields, solved the theoretical problem of defining "phase" in partially coherent light fields, and introduced the solution of non-homogeneous Neumann boundary conditions and fast discrete cosine transform in 2014.

[0007] In order to obtain as many intensity maps of different focal planes as possible to obtain a more accurate phase, the researchers proposed a strategy to use angular spectrum theory to generate a series of theoretical focal plane intensity maps through computational simulation. This method can theoretically provide different focal plane data, which helps the accuracy of phase estimation. However, compared with single-frame acquisition, this method may take more time to generate data in actual operation. In addition, since the simulation process relies on assumptions about the optical system and imaging environment, there may be certain differences between the generated defocused image and the actual captured image. This difference may be due to the simplification of the optical imaging system model, parameter uncertainty, and non-ideal factors in the actual imaging process.

[0008] Although theoretical solutions are constantly improving, the improvement of experimental equipment is also a hot topic of research. In order to improve the data acquisition speed and reduce errors, scholars proposed the concept of single phase recovery. In 2012, Almoro et al. inserted speckle field illumination on the basis of the 4f system and proposed a new solution system. In 2013, Zuo et al. used a phase spatial light modulator to modulate the defocus distance. In 2016, Tian et al. used a binocular microscope to collect different defocus images from two eyepieces to achieve phase recovery.

[0009] These methods have made remarkable progress in improving the efficiency and accuracy of phase retrieval, but some of them still suffer from problems such as complex computations, difficult to meet a priori conditions, or excessive requirements for experimental setups.

[0010] In the 1990s, Chinese scholars Yang et al. developed a phase recovery algorithm, the Yang-Gu algorithm, which uses a focal plane intensity image and a defocused intensity image to recover the phase information on the input image plane. Although this method has made a breakthrough in efficiency, the uncertainty of its iterative process and its sensitivity to noise limit its application. In order to solve these problems, domestic researchers have proposed a variety of improved algorithms, such as increasing the number of defocused images to improve the algorithm's anti-noise performance. In addition, the iterative angular spectrum algorithm (IASA) has also shown its effectiveness in phase recovery. It calculates phase information by repeatedly iterating the plane angular spectrum propagation formula between the object plane and the image plane. In 2013, researchers such as Peng Jinmang, Du Shaojun and Jiang Pengzhi from the National University of Defense Technology optimized the GS algorithm. By applying a weighted strategy at the input and output of the algorithm, the accuracy and stability of phase recovery were effectively improved, which promoted the development of imaging technology. However, the imaging speed of this method is limited to the algorithm iteration speed, and there is no noise reduction processing from the light source, which makes it difficult to achieve rapid monitoring of biological processes.

[0011] Although some systems can now capture two images in a single frame, if multiple focal planes are captured in a single frame, the axial intensity derivative can be estimated by finite difference to make the recovered phase quality higher. In 2015, Zuo et al. proposed a single-exposure quantitative phase microscopy system, but the system has certain limitations in design. Specifically, since the imaging light paths of different focal planes do not pass through the same center position of the imaging lens, this leads to the generation of aberrations, which in turn affects the imaging quality. In view of this, in actual biomedical applications, researchers are still seeking a phase recovery method with simple structure and excellent imaging quality. Summary of the invention

[0012] The purpose of the present invention is to provide a single-exposure high-order non-interference quantitative phase imaging system and method to solve the problems mentioned in the background technology.

[0013] To achieve the above-mentioned purpose, the present invention provides a single-exposure high-order non-interference quantitative phase imaging system, comprising a microscope module and a spectroscopic modulation module arranged in sequence, the microscope module comprising a halogen lamp, a condenser, an annular aperture, a sample, an objective lens, a phase plate, a lens one, a reflector and a lens two arranged in sequence along the propagation direction of the light beam, and the spectroscopic modulation module comprising a lens three, a spectroscopic module one, a spectroscopic module two, a spatial light modulator, a lens four and a CMOS camera arranged in sequence along the propagation direction of the light beam.

[0014] Preferably, the annular aperture is arranged at the rear pupil above the condenser.

[0015] Preferably, lens 1 and lens 2 form a first group 4f system, with focal lengths of f1 and f2 respectively, and lens 3 and lens 4 form a second group 4f system, with focal lengths of f3 and f4 respectively.

[0016] Preferably, the phase plate is mounted at the rear focal plane of the objective lens, a metal thin film coating is provided on the phase plate, and the size of the phase plate matches the inner diameter of the annular aperture and is optically conjugate therewith.

[0017] Preferably, the splitting module 1 includes a non-polarizing splitting prism 1 and a right-angle prism reflector 1, and the right-angle prism reflector 1 is placed above the non-polarizing splitting prism 1. The splitting module 2 includes a non-polarizing splitting prism 2 and a right-angle prism reflector 2, and the right-angle prism reflector 2 is placed on one side of the non-polarizing splitting prism 2.

[0018] Preferably, the silver-plated surfaces of the right-angle prism reflector 1 and the right-angle prism reflector 2 can both achieve a reflectivity of more than 99.5%.

[0019] The present invention also provides a single-exposure high-order non-interference quantitative phase imaging method, comprising the following steps:

[0020] S1, the light beam from the halogen lamp passes through the condenser, annular aperture, sample and phase plate in sequence;

[0021] S2, after the light beam passes through the phase plate, it is continuously split by the splitter module 1 and the splitter module 2 to split the incident light into four beams carrying the same information;

[0022] S3, four beams of light pass through a spatial light modulator, four Fresnel lens patterns with different defocus effects are loaded on the spatial light modulator, and the spatial light modulator is image-aligned and the defocus amount is adjusted;

[0023] S4. Four beams of light hit the CMOS camera with different focal points, and the CMOS camera performs phase recovery.

[0024] Preferably, the image registration in step S3 comprises the following steps:

[0025] Identify important feature points in the image, including corners and edges;

[0026] Find the feature descriptor near the feature point, which is based on the gray intensity distribution information of the local image area;

[0027] By comparing the feature points and their characteristic descriptors in the images, the similarity transformation relationship between the two images is found and the corresponding affine transformation matrix is ​​obtained;

[0028] Affine transformation matrix is ​​used to describe the geometric transformation between images to obtain a geometric transformation model. The geometric transformation includes translation, rotation and scaling.

[0029] Align the images to the same reference coordinate system based on a geometric transformation model.

[0030] Preferably, the adjusting of the defocus amount in step S3 comprises the following steps:

[0031] Calculate the path difference: Assume that the path difference of the first beam is 0; the second beam is located at a vertically upward position of the first beam, and its path difference is X1+Y1; the third beam is located at a vertically right position of the first beam, and its path difference is X2+Y2; then the fourth beam is located at a vertically upward position of the third beam, and its path difference is X1+Y1+X2+Y2; X1, Y1, X2, Y2 are the distances of light propagation, and are also the path differences caused by the parallel beam splitting of the beam splitter and the right-angle prism reflector combination;

[0032] The defocus amount generated is calculated based on the path difference: a corresponding model is made based on lens 4 of the second group 4f system, and v1=v2=v3=v4=f4 is obtained, where v1, v2, v3 and v4 respectively represent the distances between the object plane and the image plane when the imaging lenses are placed at any position with equal spacing; the image plane generated by lens 4 moves a corresponding distance according to the position where lens 4 moves, and the defocus amount generated is obtained : , where i represents the i-th light beam;

[0033] Calculate the defocus effect of equal spacing: Assuming that the first image is the in-focus image and corresponds to the first beam of light, the defocus effect of the first Fresnel lens pattern loaded is ; Assume that the second picture has a defocus value of , and corresponds to the second beam of light, then the defocus effect of the second Fresnel lens pattern loaded is - ; Assume that the third picture has a defocus value of , and corresponds to the third beam of light, then the defocus effect of the third Fresnel lens pattern loaded is ; Assume that the fourth picture has a defocus value of 3 , and corresponds to the fourth beam of light, then the defocus effect of the fourth Fresnel lens pattern loaded is ;

[0034] By changing the angular spectrum transfer function pattern displayed on the spatial light modulator, the four Fresnel lens patterns with different defocus amounts loaded on the spatial light modulator are compensated. The formula is:

[0035] ;

[0036] in, is the spatial coordinate of the plane where the light beam hits the liquid crystal loading pattern area of ​​the SLM, is the wavelength of light, is the wave number, Is an imaginary unit.

[0037] Preferably, the phase recovery in step S4 includes the following steps:

[0038] A CMOS camera is used to collect a light intensity image of a sample, and the light intensity image is divided into four areas: top, bottom, left, and right. The four areas are composed of the focal plane and three images with , , 3 The sub-images are composed of the out-of-focus distance effect;

[0039] Taking the center of the intensity image as the reference point, it is divided into four sub-images according to the top, bottom, left and right directions. The four sub-images are registered to ensure that the sizes are completely consistent. After processing, four intensity images of focus and defocus are finally obtained, which are I(x, y), I △z (x,y),I 2△z (x,y) and I 3△z (x,y);

[0040] The collected focused and defocused light intensity images are numerically differentiated in high order to obtain the axial differential of light intensity with an accuracy order of 3. ;

[0041] ;

[0042] Among them, I0, I1, I2 and I3 represent the collected intensity images I(x, y), I △z (x,y),I 2△z (x,y) and I 3△z (x,y);

[0043] According to the axial differentiation of light intensity and the focused light intensity image I(x,y), and the phase is obtained based on the intensity transfer equation :

[0044] ;

[0045] in, is the inverse Laplace operator, is the gradient operator, is the vector dot product, and All need to be realized through Fourier transform, and the Fourier transform formula is as follows:

[0046] ;

[0047] in, F represents the Fourier transform, and represents the inverse Fourier transform, ( , ) is the spatial coordinate ( x , y ) corresponding frequency domain coordinates;

[0048] The physical thickness H(x,y) of the sample is obtained by conversion using the following formula:

[0049] ;

[0050] in, n is the difference in refractive index between the sample and the surrounding medium;

[0051] Using the four image data, the finite difference method is used to estimate the first-order derivative of the axial intensity, and the error of the axial intensity estimation on two planes is analyzed to obtain the first-order derivative estimate :

[0052] ;

[0053] Substitute the improved estimate into the phase calculation formula to solve the high-precision phase, then select a set of images, and by weighting their combination, eliminate the high-order terms effectively, and only keep the first-order terms. Based on the linear differential theory, the selected weighted images are superimposed to obtain a new phase estimate.

[0054] ;

[0055] in, represents the weighting coefficient of the image, and I j Indicates the degree of defocus The intensity image is obtained at the position, I0 is the focal plane image, I j Indicates a defocused image, I j Represents an overfocus image, and the total number of images is m+n+1=4.

[0056] Therefore, the present invention adopts the above-mentioned single-exposure high-order non-interference quantitative phase imaging system and method, which has the following beneficial effects: retaining the advantages of the white light illumination system such as stable structure, insensitivity to environmental interference, high phase sensitivity and low spatial noise, and at the same time, through parallel splitting technology and SLM loading four Fresnel lens patterns with different defocus amounts, to compensate for the low time resolution of the system, low axial derivative estimation accuracy and unequal spacing path difference introduced by the prism; constructing a single-frame multi-focal plane phase recovery microscopy imaging platform to achieve high-resolution, high-sensitivity real-time monitoring and quantitative analysis of cells; using two sets of photodetector modules to divide the incident light into four beams carrying the same information, and introducing different optical path differences respectively, so that the camera can simultaneously obtain four light intensity images of different focal planes with a single sampling; proposing a method based on The image registration method of intensity is used to align the spatial position rotation and difference of the four-window images caused by the mechanical deviation of the optical path, so as to realize the accurate recovery of the phase; the single-exposure high-order phase recovery system collects four different focal plane images, and the imaging accuracy is improved by two orders compared with the system that collects two different focal plane images in a single frame, which makes up for the fact that the traditional spectroscopic technology cannot obtain four different focal planes; a method of generating four different Fresnel lens modulation patterns on an SLM liquid crystal plane at the same time is proposed and developed for free focusing; the intensity differential is solved by using four different focal plane intensity images and combined with the phase recovery algorithm of the fast Fourier transform method to solve the TIE recovery phase, which not only ensures the imaging speed but also makes the axial intensity differential estimation more accurate; on the basis of ensuring the imaging speed, the accuracy of phase recovery is significantly improved.

[0057] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 It is a structural schematic diagram of a single-exposure high-order non-interference quantitative phase imaging system according to an embodiment of the present invention;

[0059] Figure 2 Three views of a light splitting module 1 according to an embodiment of the present invention, (a) is a front view, (b) is a top view, and (c) is a right view;

[0060] Figure 3 Three views of the light splitting module 2 according to an embodiment of the present invention, (a) is a front view, (b) is a top view, and (c) is a right view;

[0061] Figure 4 Four Fresnel lens patterns with different defocus amounts are simultaneously loaded on the spatial light modulator of the embodiment of the present invention;

[0062] Figure 5Schematic diagram of the outgoing light path of the light splitting module 2 according to an embodiment of the present invention, ① is the first light beam, ② is the second light beam, ③ is the third light beam, and ④ is the fourth light beam;

[0063] Figure 6 A schematic diagram of the image plane movement of the imaging lens caused by the distance difference according to an embodiment of the present invention;

[0064] Reference numerals

[0065] 1. Microscope module; 2. Spectral modulation module; 3. Halogen lamp; 4. Condenser; 5. Annular aperture; 6. Sample; 7. Objective lens; 8. Phase plate; 9. Lens 1; 10. Reflector; 11. Lens 2; 12. Lens 3; 13. Spectral module 1; 14. Spectral module 2; 15. Spatial light modulator; 16. Lens 4; 17. CMOS camera. DETAILED DESCRIPTION

[0066] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0067] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0068] Example

[0069] like Figure 1 As shown, the present invention provides a single-exposure high-order non-interference quantitative phase imaging system, comprising a microscope module 1 and a spectroscopic modulation module 2 arranged in sequence. The microscope module 1 comprises a halogen lamp 3, a condenser 4, an annular diaphragm 5, a sample 6, an objective lens 7, a phase plate 8, a lens 1 9, a reflector 10 and a lens 2 11 arranged in sequence along the propagation direction of the light beam. The annular diaphragm 5 is arranged at the rear pupil above the condenser 4, the phase plate 8 is installed at the rear focal plane of the objective lens 7, a metal film coating is arranged on the phase plate, and the size of the phase plate 8 matches the inner diameter of the annular diaphragm 5 and is optically conjugate therewith. The lens 1 9 and the lens 2 11 form a first group of 4f systems, with focal lengths of f1 and f2 respectively.

[0070] The light splitting modulation module 2 includes a lens 3 12, a light splitting module 1 13, a light splitting module 2 14, a spatial light modulator 15, a lens 4 16 and a CMOS camera 17 arranged in sequence along the light beam propagation direction. The lens 3 12 and the lens 4 16 form a second group 4f system, with focal lengths of f3 and f4 respectively.

[0071] Reference Figure 2 , the light splitting module 13 includes a non-polarized light splitting prism 1 and a right-angle prism reflector 1, and the right-angle prism reflector 1 is placed above the non-polarized light splitting prism 1. Figure 3 The light splitting module 2 14 includes a non-polarized light splitting prism 2 and a right angle prism reflector 2, and the right angle prism reflector 2 is placed on one side of the non-polarized light splitting prism 2. The silver-plated surfaces of the right angle prism reflector 1 and the right angle prism reflector 2 can both achieve a reflectivity of more than 99.5%.

[0072] The imaging system adopts the Kohler illumination method and uses the halogen lamp 3 as the light source to overcome the speckle noise caused by the laser. Using the Kohler illumination method, an annular aperture 5 is placed at the rear pupil above the condenser 4 to suppress the background light intensity and ensure that the extremely weak light intensity that is transmitted through the sample 6 and scattered by the sample 6 is strong enough. A phase plate 8 is installed on the rear focal plane of the objective lens 7. The phase plate 8 matches the inner diameter of the annular aperture 5 and is optically conjugate with it. The phase difference of the surrounding light relative to the diffracted light is changed by π / 2. At the same time, the metal film coating on the phase plate 8 can reduce the amplitude of the surrounding light by 60%-90%. The contrast between the light intensity of the surrounding light and the diffracted light directly determines the contrast of the final image.

[0073] The present invention also provides a single-exposure high-order non-interference quantitative phase imaging method, comprising the following steps:

[0074] S1, the light beam from the halogen lamp passes through the condenser, annular aperture, sample and phase plate in sequence;

[0075] S2, after the light beam passes through the phase plate, it is continuously split by the splitter module 1 and the splitter module 2 to split the incident light into four beams carrying the same information;

[0076] S3, four beams of light pass through the spatial light modulator, four Fresnel lens patterns with different defocus effects are loaded on the spatial light modulator (SLM), and the spatial light modulator is image-aligned and the defocus amount is adjusted;

[0077] S4. Four beams of light hit the CMOS camera with different focal points, and the CMOS camera performs phase recovery.

[0078] The light carrying the sample information after passing through the phase plate is continuously split by the splitter module 1 and the splitter module 2, dividing the incident light into four beams carrying the same information. Due to the particularity of the two right-angle prism reflectors, it is difficult to ensure that the reflected light will not bring in a small amount of path difference, which may directly cause the subsequent imaging surface to produce a defocus effect, but this defocus amount can be calculated based on the system parameters. Figure 4 In order to ensure that the defocus of the four beams is equally spaced and controllable at the same time, four Fresnel lens patterns with different defocus effects are loaded on the spatial light modulator, so that the four beams of light hit the CMOS camera at different focal points, introducing different path differences for the four beams of light. The light transmitted from the surface of the spatial light modulator is Fourier transformed by lens 4 and captured by the CMOS camera. At this time, the camera simultaneously acquires three equally spaced focus images and one image on the focal plane with a single sampling.

[0079] After initial exploration and verification, it was found that the intensity difference between the four images caused by the combined light splitting of NPBS and right-angle prism reflector theoretically does not exceed 0.5%, so there is basically no need to consider the impact of the intensity difference. However, for the process of recovering the phase by the intensity transfer equation, the control of the defocus amount is very important. Although the distance difference can be reduced by moving the relative positions of the NPBS and the right-angle prism reflector, it is still difficult to achieve equal distance focal planes through this mechanical movement. At the same time, due to the mechanical deviation of the non-polarizing beam splitter (NPBS) and the right-angle prism reflector, the four window images collected on the CMOS camera will rotate and differ in spatial position, which will cause difficulties in the subsequent phase recovery and image processing process. Therefore, it is necessary to calibrate the spatial light modulator or align the images, and achieve the effect of the same CMOS camera collecting four frames of images in chronological order by aligning the images of the four windows.

[0080] The image registration in step S3 is mainly to register the feature signals in the image, and to achieve four-window image registration under the combined spectroscopy of the two spectroscopy modules by adjusting the accuracy and stability of feature point matching, which specifically includes the following steps:

[0081] Identify important feature points in the image, which can be corners, edges, etc.

[0082] Find the feature descriptors near the feature points, which are based on the gray intensity distribution information of the local image area;

[0083] By comparing the feature points and their characteristic descriptors in the images, the similarity transformation relationship between the two images, that is, the corresponding affine transformation matrix, is found;

[0084] Affine transformation matrix is ​​used to describe geometric transformations such as translation, rotation and scaling between images to obtain a geometric transformation model;

[0085] Based on the estimated geometric transformation model, the images are aligned to the same reference coordinate system.

[0086] Different from traditional methods such as Scale Invariant Feature Transform (SIFT) or Speeded Up Robust Features (SURF), the image registration method in this scheme focuses more on using grayscale information to match features between different images and establish correspondence between them. This method is not only applicable to image registration of the same imaging modality, but also can handle registration problems between different imaging modalities, thereby improving the accuracy and robustness of image registration in a variety of application scenarios.

[0087] The step S3 of adjusting the defocus amount includes the following steps:

[0088] Calculate the distance difference: refer to Figure 5 , assuming that the path difference of the first beam is 0 (L1=0); the second beam is located at the vertical upward position of the first beam, and its path difference is X1+Y1 (L2=X1+Y1); the third beam is located at the vertical right position of the first beam, and its path difference is X2+Y2 (L3=X2+Y2); then the fourth beam is located at the vertical upward position of the third beam, and its path difference is X1+Y1+X2+Y2 (L4=X1+Y1+X2+Y2). Reference Figure 2-3 , X1, Y1, X2, Y2 are the distances that light travels, and are also the path differences caused by the parallel beam splitting of the combination of the beam splitter prism and the right-angle prism reflector.

[0089] Calculate the defocus amount based on the path difference: When the four beams are split in parallel, different path differences are introduced respectively, which is equivalent to adding a distance (path difference) to the two lenses in the second group of 4f system. At this time, the optical path is analyzed as an ideal optical system: refer to Figure 6 , according to the lens 4 of the second group of 4f system, make a corresponding model, v1=v2=v3=v4=f4, where v1, v2, v3 and v4 respectively represent the distance between the object plane and the image plane when the imaging lens is placed at any position with equal spacing. It can be seen from this that if a path difference is introduced in the second group of 4f system, it is equivalent to moving lens 4 backwards, but this will not affect its focal length. Therefore, the image plane produced by lens 4 will also move a corresponding distance due to the position of lens 4, that is, the defocus amount produced is: .

[0090] Calculate the defocus effect of equal spacing: Assuming that the first image is the in-focus image and corresponds to the first beam of light, the defocus effect of the first Fresnel lens pattern loaded is ; Assume that the second picture has a defocus value of , and corresponds to the second beam of light, then the defocus effect of the second Fresnel lens pattern loaded is - ; Assume that the third picture has a defocus value of , and corresponds to the third beam of light, then the defocus effect of the third Fresnel lens pattern loaded is ; Assume that the fourth picture has a defocus value of 3 , and corresponds to the fourth beam of light, then the defocus effect of the fourth Fresnel lens pattern loaded is .

[0091] By changing the angular spectrum transfer function displayed on the spatial light modulator Pattern, four Fresnel lens patterns with different defocus amounts loaded on the spatial light modulator are compensated to change the angular spectrum transfer function as follows:

[0092] ;

[0093] in, is the spatial coordinate of the plane where the light beam hits the liquid crystal loading pattern area of ​​the SLM, is the wavelength of light, is the wave number, Is an imaginary unit.

[0094] The phase recovery in step S4 includes the following steps:

[0095] A CMOS camera is used to collect a light intensity image of a sample, and then the image is divided into four areas: top, bottom, left, and right. The four areas are composed of the focal plane and three images with , , 3 The sub-images are composed of the defocus distance effect;

[0096] Directly divide the captured image into four parts with the middle as the reference point, and cut it into I(x, y), I △z (x,y),I 2△z (x,y) and I 3△z (x, y) four sub-images, image registration is performed on the four sub-images, and the sizes are ensured to be completely consistent. After processing, four focused and defocused light intensity images are finally obtained;

[0097] Referring to Table 1, the collected focused and defocused light intensity images I(x, y), I △z (x,y),I 2△z (x,y) and I 3△z (x, y) is numerically differentiated in high order according to formula (2) to obtain the axial differential of the light intensity with an accuracy order of 3. ;

[0098] ;

[0099] Among them, I0, I1, I2 and I3 represent the collected intensity images I(x, y), I △z (x,y),I 2△z (x,y) and I 3△z (x,y).

[0100] According to the axial differentiation of light intensity The phase is obtained by solving the intensity transfer equation based on the focused light intensity image I(x,y) by the following formula: :

[0101] ;

[0102] in, is the inverse Laplace operator, is the gradient operator, is the vector dot product, where and All operators need to be implemented through Fourier transform. The Fourier transform formula is as follows:

[0103] ;

[0104] in, F represents the Fourier transform, and represents the inverse Fourier transform, ( , ) is the spatial coordinate ( x , y ) The frequency domain coordinates corresponding to the sample; of course, if the physical thickness of the sample needs to be obtained, the physical thickness of the sample H(x,y) can be obtained by the following formula:

[0105] ;

[0106] in, n is the difference in refractive index between the sample and the surrounding medium.

[0107] In order to improve the accuracy of the phase retrieval technique based on the transmission equation and reduce noise interference, a high-precision estimation method is used. This method uses the four image data and the finite difference method to estimate the first-order derivative of the axial intensity. The first-order derivative estimation formula is obtained, and the purpose is to measure small changes in intensity in the focus plane at z=0. Considering that the propagation of light in small defocused areas is not linear, a new linear differential measurement method is introduced to replace the traditional finite difference approximation to accurately estimate and correct high-order differential errors caused by nonlinear effects. First-order derivative estimation The formula is as follows:

[0108] ;

[0109] In order to obtain a more accurate estimate of the first-order derivative, the errors in the axial intensity estimates on the two planes are analyzed. These errors are mainly caused by high-order differentials, as shown in the brackets in formula (6); in order to optimize the estimate of the first-order derivative, it is necessary to subtract the errors caused by this part of the high-order differential as much as possible; then, this improved estimate is substituted into formula (3) to solve the high-precision phase. The finite difference method can quantify and eliminate the influence of high-order differentials. At this time, a group of images are selected, and by weighting their combination, the high-order terms in formula (6) are effectively eliminated, leaving only the first-order terms. Based on the theory of linear differentials, the selected weighted images are superimposed to obtain a more accurate phase estimate.

[0110] ;

[0111] in, represents the weighting coefficient of the image, and I j Indicates the degree of defocus Here, I0 is the focal plane image, I j Indicates a defocused image, I j represents an overfocus image. Therefore, the total number of images is m+n+1=4. The weighting coefficients for each image are determined to eliminate the unnecessary high-order differential terms. By solving these conditions, a set of image weighting coefficients are obtained that can ensure the required order accuracy, thereby constructing the following image weighting table:

[0112] Table 1 Image weighting table with different phase order accuracy

[0113] .

[0114] In this method, m+n=3 represents the differential order to be corrected. In summary, the present invention adopts an intensity differential method combining four intensity images with different focal lengths and combines it with a fast Fourier transform (FFT) phase recovery method. This method effectively improves the accuracy and robustness of phase recovery by utilizing intensity information at different focal lengths.

[0115] Therefore, the present invention adopts the above-mentioned single-exposure high-order non-interference quantitative phase imaging system and method, through the annular light illumination optical path, parallel splitting technology, SLM loaded with Fresnel lenses of different focal lengths and high-order intensity transfer equation to restore the phase algorithm, while ensuring the imaging quality and imaging field of view, the imaging accuracy is improved by two orders relative to the phase obtained from the two light intensity images.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A single-exposure high-order non-interference quantitative phase imaging system, characterized by: The invention comprises a microscope module and a spectroscopic modulation module which are arranged in sequence, wherein the microscope module comprises a halogen lamp, a condenser, an annular aperture, a sample, an objective lens, a phase plate, a lens 1, a reflector and a lens 2 which are arranged in sequence along the propagation direction of the light beam, and the spectroscopic modulation module comprises a lens 3, a spectroscopic module 1, a spectroscopic module 2, a spatial light modulator, a lens 4 and a CMOS camera which are arranged in sequence along the propagation direction of the light beam; After the light beam passes through the phase plate, it is continuously split by the splitter module 1 and the splitter module 2, and the incident light is divided into four beams of light carrying the same information; the four beams of light pass through the spatial light modulator, and four Fresnel lens patterns with different defocus effects are loaded on the spatial light modulator, and the spatial light modulator is image-aligned and the defocus amount is adjusted; The annular aperture is set at the rear pupil above the condenser; The phase plate is installed at the rear focal plane of the objective lens, and a metal thin film coating is arranged on the phase plate. The size of the phase plate matches the inner diameter of the annular aperture and is optically conjugate therewith; The first light splitting module includes a non-polarizing light splitting prism and a right-angle prism reflector. The right-angle prism reflector is placed above the first non-polarizing light splitting prism. The second light splitting module includes a non-polarizing light splitting prism and a right-angle prism reflector. The right-angle prism reflector is placed on one side of the second non-polarizing light splitting prism.

2. The single-exposure high-order non-interference quantitative phase imaging system according to claim 1, characterized in that: Lens 1 and lens 2 form the first 4f system, with focal lengths of f1 and f2 respectively. Lens 3 and lens 4 form the second 4f system, with focal lengths of f3 and f4 respectively.

3. The single-exposure high-order non-interference quantitative phase imaging system according to claim 1, characterized in that: The silver-plated surfaces of the right-angle prism reflector 1 and the right-angle prism reflector 2 can both achieve a reflectivity of more than 99.5%.

4. A single-exposure high-order non-interference quantitative phase imaging method, applied to a single-exposure high-order non-interference quantitative phase imaging system as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: S1, the light beam from the halogen lamp passes through the condenser, annular aperture, sample and phase plate in sequence; S2, after the light beam passes through the phase plate, it is continuously split by the splitter module 1 and the splitter module 2 to split the incident light into four beams carrying the same information; S3, four beams of light pass through a spatial light modulator, four Fresnel lens patterns with different defocus effects are loaded on the spatial light modulator, and the spatial light modulator is image-aligned and the defocus amount is adjusted; S4. Four beams of light hit the CMOS camera with different focal points, and the CMOS camera performs phase recovery.

5. The single-exposure high-order non-interference quantitative phase imaging method according to claim 4, characterized in that: The image registration in step S3 includes the following steps: Identify important feature points in the image, including corners and edges; Find the feature descriptor near the feature point, which is based on the gray intensity distribution information of the local image area; By comparing the feature points and their characteristic descriptors in the images, the similarity transformation relationship between the two images is found and the corresponding affine transformation matrix is ​​obtained; Affine transformation matrix is ​​used to describe the geometric transformation between images to obtain a geometric transformation model. The geometric transformation includes translation, rotation and scaling. Align the images to the same reference coordinate system based on a geometric transformation model.

6. The single-exposure high-order non-interference quantitative phase imaging method according to claim 5, characterized in that: The step S3 of adjusting the defocus amount includes the following steps: Calculate the path difference: Assume that the path difference of the first beam is 0; the second beam is located at a vertically upward position of the first beam, and its path difference is X1+Y1; the third beam is located at a vertically right position of the first beam, and its path difference is X2+Y2; then the fourth beam is located at a vertically upward position of the third beam, and its path difference is X1+Y1+X2+Y2; X1, Y1, X2, Y2 are the distances of light propagation, and are also the path differences caused by the parallel beam splitting of the beam splitter and the right-angle prism reflector combination; The defocus amount generated is calculated based on the path difference: a corresponding model is made based on lens 4 of the second group 4f system, and v1=v2=v3=v4=f4 is obtained, where v1, v2, v3 and v4 respectively represent the distances between the object plane and the image plane when the imaging lenses are placed at any position with equal spacing; the image plane generated by lens 4 moves a corresponding distance according to the position where lens 4 moves, and the defocus amount generated is obtained : , where i represents the i-th light beam; Calculate the defocus effect of equal spacing: Assuming that the first image is the in-focus image and corresponds to the first beam of light, the defocus effect of the first Fresnel lens pattern loaded is ; Assume that the second picture has a defocus value of , and corresponds to the second beam of light, then the defocus effect of the second Fresnel lens pattern loaded is ; Assume that the third picture has a defocus value of , and corresponds to the third beam of light, then the defocus effect of the third Fresnel lens pattern loaded is ; Assume that the fourth picture has a defocus value of , and corresponds to the fourth beam of light, then the defocus effect of the fourth Fresnel lens pattern loaded is ; By changing the angular spectrum transfer function pattern displayed on the spatial light modulator, the four Fresnel lens patterns with different defocus amounts loaded on the spatial light modulator are compensated. The formula is: ； in, is the spatial coordinate of the plane where the light beam hits the liquid crystal loading pattern area of ​​the SLM, is the wavelength of light, is the wave number, Is an imaginary unit.

7. The single-exposure high-order non-interference quantitative phase imaging method according to claim 6, characterized in that: The phase recovery in step S4 includes the following steps: A CMOS camera is used to collect a light intensity image of a sample, and the light intensity image is divided into four areas: top, bottom, left, and right. The four areas are composed of the focal plane and three images with , , The sub-images are composed of the out-of-focus distance effect; Taking the center of the intensity image as the reference point, it is divided into four sub-images according to the top, bottom, left and right directions. The four sub-images are registered to ensure that the sizes of the four sub-images are completely consistent. Then, four intensity images of focus and defocus are obtained, which are I(x, y), I(x, y), and I(x, y). △z (x,y),I 2△z (x,y) and I 3△z (x,y); The collected focused and defocused light intensity images are numerically differentiated in high order to obtain the axial differential of light intensity with an accuracy order of 3. ; ； Among them, I0, I1, I2 and I3 represent the collected intensity images I(x, y), I △z (x,y),I 2△z (x,y) and I 3△z (x,y); According to the axial differentiation of light intensity and the focused light intensity image I(x,y), and the phase is obtained based on the intensity transfer equation : ； in, is the inverse Laplace operator, is the gradient operator, is the vector dot product, and All need to be realized through Fourier transform, and the Fourier transform formula is as follows: ； in, F represents the Fourier transform, and represents the inverse Fourier transform, ( , ) is the spatial coordinate The corresponding frequency domain coordinates; The physical thickness H(x,y) of the sample is obtained by conversion using the following formula: ； in, is the difference in refractive index between the sample and the surrounding medium; Using the four image data, the finite difference method is used to estimate the first-order derivative of the axial intensity, and the error of the axial intensity estimation on two planes is analyzed to obtain the first-order derivative estimate : ； Substitute the improved estimate into the phase calculation formula to solve the high-precision phase, then select a set of images and weight their combination to effectively eliminate the high-order terms, retaining only the first-order terms. Based on the linear differential theory, the selected weighted images are superimposed to obtain a new phase estimate. ； in, represents the weighting coefficient of the image, and I j Indicates the degree of defocus The intensity image is obtained at the position, I0 is the focal plane image, I j represents an out-of-focus image, I j Represents an overfocus image, and the total number of images is m+n+1=4.

Images