CN106768396A - A kind of method and system based on differential contrast imaging reduction quantitative phase images - Google Patents

Abstract

The invention discloses a kind of method and system based on differential contrast image Quantitative reduction phase image, it is related to computer generated image field.The present invention establishes the target image of stabilization to the method for reduction quantitative phase images, and the present invention initially sets up differential contrast and is imaged two-dimension optical phase transfer function H (u), and then sets up H (u), the frequency-domain function of differential contrast imageAnd the relation between quantitative phase information, it is final to realize that carrying out de-convolution operation by the phase transfer function being imaged to differential contrast recovers quantitative phase information, further the present invention have studied multiaxis and divide the method to form Quantitative reduction phase information under asymmetric lighting pattern to strengthen the re-configurability of phase information on different directions, and use mathematics to optimize to reduce the increased problem of frequency noise caused by direct deconvolution.Instant invention overcomes the defect that traditional quantitative image acquisition operations are complicated, image-forming condition is harsh, and the image resolution ratio for obtaining is higher.

Description

A method and system for restoring quantitative phase images based on differential phase contrast imaging

technical field

The invention relates to the technical field of phase imaging, in particular to a method for restoring a quantitative phase image based on a differential phase contrast image and a quantitative phase reduction microscopic imaging system based on an LED array light source.

Background technique

In some specific fields, such as optical measurement, material physics, adaptive optics, X-ray diffraction optics, electron microscopy, biomedical imaging, etc., most samples are phase objects. The amplitude transmittance distribution of such objects is uniform, but the spatial distribution of refractive index or thickness is not uniform, so the light wave amplitude of the phase object changes very little, but the phase change is very large. The human eye or other photodetectors can only judge the change of the amplitude of the object but not the change of its phase, so they cannot distinguish the parts with different thickness or refractive index in the phase object. Therefore, for these fields, it is particularly important to obtain phase information.

Phase contrast (Phase Contrast) microscopy technology is a milestone in phase imaging technology. Its basic principle is to separate the background light of illumination from the scattered light of the sample, so as to reveal the details of the foreground, so that the phase change is presented in the form of intensity change. However, traditional phase contrast microscopy has disadvantages in terms of phase inversion, halo and fading effects, sample thickness, influence of cover glass and slide glass, etc. In order to solve these problems, a series of new technologies such as differential interference contrast imaging (DIC) and differential phase contrast imaging (DPC) have appeared successively on the basis of phase contrast microscopy. However, these techniques have drawbacks such as complex operations and harsh imaging conditions. More importantly, these methods cannot obtain global quantitative phase information.

Quantitative phase imaging visualizes and detects the refractive properties of a sample by measuring the phase delay of a beam of light. In optical measurement, due to the high frequency of light waves, detectors cannot directly detect the phase distribution of light. In actual operation, the data that can be directly measured is often only the intensity distribution of the wave field. Therefore, phase recovery is an important topic in optical measurement and imaging technology. How to obtain phase information from intensity measurement data has become a technical problem to be solved in research.

Contents of the invention

In view of the foregoing, the present invention proposes a quantitative phase restoration microscopic imaging system and a method for restoring quantitative phase images based on differential phase contrast images in order to solve the above technical problems.

For this reason, the present invention adopts following technical scheme:

On the one hand, the present invention provides a quantitative phase reduction microscopic imaging system, including a transmission microscope, characterized in that the light source of the transmission microscope is an LED array, and the illumination pattern of the LED array is controlled by programming, and also includes a The multi-dimensional displacement platform for the position of the LED array light source, the camera equipment for collecting the observation results of the microscope, and the processing equipment for restoring the collected images to quantitative phase images.

Furthermore, the LED array of the present invention is a rectangular array composed of m×n light-emitting diodes arranged at equal intervals, where m and n are both positive integers; the lighting pattern of the LED array adopts a static screen mode.

Further, the present invention also includes a control device, and the controller is respectively connected with the LED array light source and the photographing device to realize switching of lighting patterns and synchronizing with camera acquisition frequency by using a time-division multiplexing mode; specifically, the time-division multiplexing mode refers to A single control device can complete the signal triggering of LED array lighting pattern switching and the triggering of camera acquisition signals in different time periods, which can save control pin resources. Since the trigger time is extremely short, it should not affect the synchronization of pattern switching and camera acquisition . Furthermore, the processing device of the present invention can complete the export of image data and the calculation and processing of image data, and finally obtain the quantitative phase imaging picture of the sample; specifically, the embodiment of the present invention uses software that matches the camera device to complete the triggering and processing of image acquisition. For image export, use image processing tools such as MATLAB or VC++ with the same processing effect to complete the calculation of image data;

The specific processing method of processing equipment of the present invention is as follows:

Step A: Obtain the differential phase contrast imaging picture I _DPC divided by any symmetry axis direction of the sample; define the first target image collected under the first differential illumination as I ₁ , and define the second target image under the second differential illumination as I ₂ , then the method to obtain _IDPC is:

Step B: Establish the two-dimensional optical phase transfer function H(u) of differential phase contrast imaging based on the weak target transfer function, and its expression is as follows:

H(u)=i[∫∫S(u′)P*(u′)p(u′+u)d ² u′-∫∫s(u′)P*(u′)p(u′- u)d ² u′]

Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane;

Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows:

Then the quantitative phase information restored in the Fourier domain is:

right The inverse Fourier transform finally obtains quantitative phase information.

Since the above differential phase contrast imaging result is a differential phase contrast image acquired in the direction of a single axis of symmetry, it does not contain phase information perpendicular to the axis of symmetry; in order to enhance the ability of phase reconstruction in different directions, multi-axis imaging can be used along any direction Divide and form an asymmetric illumination pattern, so as to obtain differential phase contrast imaging pictures containing phase information in different directions, and then further complete the differential phase contrast imaging containing different phase information and restore them to quantitative phase pictures.

Based on the above, the present invention provides a processing method for obtaining quantitative phase imaging pictures of samples under the division of multiple symmetry axes to form an asymmetric illumination pattern, specifically as follows:

Step A: Obtain differential phase contrast imaging pictures divided by any two or more symmetry axis directions of the sample

Define that when the illumination pattern is divided by the first axis of symmetry, the first target image collected by the first differential illumination light is I ₁₁ , and the second target image collected by the second differential illumination light is I ₁₂ ; the definition is symmetric by the jth When the illumination pattern is further divided by the axis, the first target image collected by the first differential illumination light is I _j1 , and the second target image collected by the second differential illumination light is I _j2 ; then the differential phase contrast image is:

Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function, and its expression is as follows:

H(u)=i[∫∫s(u′)P ^* (u′)p(u′+u)d ² u′-∫∫S(u′)P ^* (u′)p(u′- u)d ² u′]

Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane;

Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows:

Solving the above equations jointly, the quantitative phase information based on multi-axis division is calculated by the following formula:

Since the frequency point at the intersection of H(u) and the axial edge is zero and the frequency involved in the operation is wide, direct deconvolution will greatly superimpose the noise on these frequencies, so the problem can be solved by the regularized least squares method, and finally The simplified quantitative phase information expression is as follows: where, α is the regularization parameter

On the other hand, the present invention provides a method for restoring a quantitative phase image based on a differential phase contrast image, wherein the division of an asymmetric illumination pattern is based on a single axis of symmetry for division according to any direction, including the following steps:

Step A: Using a program-controlled LED array as a light source, controlling the switching of the lighting pattern of the LED array light source based on the time-division multiplexing mode to synchronize with the frequency of image acquisition by the camera, wherein the target image is collected through an asymmetrical lighting pattern, and the asymmetrical The illumination pattern is divided by a single axis of symmetry; define the first target image collected under the first differential illumination as I ₁ , and the second target image under the second differential illumination as I ₂ , then the differential phase contrast image is;

Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function, and its expression is as follows:

H(u)=i[∫∫S(u′)P*(u′)p(u′+u)d ² u′-∫∫S(u′)P*(u′)p(u′- u)d ² u′]

Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane;

Step C: The model of the Fourier spectrum energy of the differential phase contrast image is established as follows:

Then the quantitative phase information restored in the Fourier domain is:

right Inverse Fourier transform is performed to finally obtain quantitative phase information.

Since the above differential phase contrast imaging result is a differential phase contrast image acquired in the direction of a single axis of symmetry, it does not contain phase information perpendicular to the axis of symmetry; in order to enhance the ability of phase reconstruction in different directions, multi-axis imaging can be used along any direction Divide and form an asymmetric illumination pattern, so as to obtain differential phase contrast imaging pictures containing phase information in different directions, and then further complete the differential phase contrast imaging containing different phase information and restore them to quantitative phase pictures.

Based on the above, the present invention provides a method for restoring a quantitative phase image based on a differential phase contrast image, wherein the division of an asymmetric illumination pattern is divided according to any direction using two or more symmetry axes, including the following steps:

Step A: Using a program-controlled LED array as the light source, controlling the lighting pattern of the LED array light source based on the time-division multiplexing mode to be synchronized with the frequency of image acquisition by the photographic equipment, wherein the target image is collected through an asymmetrical lighting pattern, and the asymmetrical lighting The pattern is divided by two or more symmetry axes in any direction; the acquisition target image is specifically operated in the following order:

A1: Use the first axis of symmetry to divide the illumination pattern, define the first target image acquired under the first differential illumination as I ₁₁ , and define the second target image acquired under the second differential illumination as I ₁₂ ;

A2: Use the jth symmetry axis to further divide the illumination pattern, define the first target image collected under the first differential illumination as I _j1 , and define the second target image collected under the second differential illumination as I _j2 ;

A3: Repeat the operation of step A2 until the target axis number is reached, then the differential phase contrast images of the two target images obtained in each step are as follows:

Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function:

H(u)=i[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′-∫∫S(u′)P ^* (u′)p(u′- u)d ² u′]

Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane;

Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows:

Solving the above equations jointly, the quantitative phase information based on multi-axis division is calculated by the following formula:

Since the frequency point at the intersection of H(u) and the axial edge is zero and the frequency involved in the operation is wide, direct deconvolution will greatly superimpose the noise on these frequencies, so the problem can be solved by the regularized least squares method, and finally The simplified quantitative phase information expression is as follows: where, α is the regularization parameter

The present invention has following beneficial effects:

The system disclosed in the present invention can restore quantitative phase information based on differential phase contrast imaging, and the present invention establishes a stable method from target image to quantitatively restored phase image, which overcomes the defects of traditional quantitative image acquisition operation complexity and harsh imaging conditions, and The obtained image has a higher resolution; the present invention builds a quantitative phase restoration microscopic imaging system based on LED array light sources, adopts asymmetrical illumination pattern synchronization and photographic equipment to acquire the target image, and then uses processing equipment for calculation and processing, and finally obtains quantitative phase information. The present invention obtains the differential phase contrast imaging picture through processing, and the differential phase contrast imaging picture contains phase gradient information. The present invention first establishes the differential phase contrast imaging two-dimensional optical phase transfer function H(u) (the specific derivation process is as described above) , and then establish H(u), the frequency domain function of the differential phase contrast image The relationship between the phase transfer function and the quantitative phase information is realized, and finally the quantitative phase information can be recovered by performing deconvolution operation on the phase transfer function of the differential phase contrast imaging. The present invention is not limited to the division of a single symmetry axis to form an asymmetric illumination pattern, further, the present invention forms an asymmetry illumination pattern by dividing a plurality of symmetry axes along any direction, and then obtains multiple differential phase contrast images containing different phase information picture, and then perform a joint solution to finally restore the quantitative phase information to enhance the reconstruction ability of phase information in different directions. In addition, the present invention also optimizes the quantitative phase information formula based on multi-axis division.

Description of drawings

1 is a schematic diagram of the imaging principle of the LED array light source microscopic imaging system according to the embodiment of the present invention;

Fig. 2 is a flow chart of using the LED array light source microscopic imaging system to restore the quantitative phase image according to the embodiment of the present invention;

3 is a schematic structural diagram of a microscopic imaging system based on an LED array light source according to an embodiment of the present invention;

Fig. 4 is a flow chart of optical path correction of the LED array light source according to the embodiment of the present invention;

Fig. 5 is a schematic diagram of an illumination pattern for phase contrast imaging according to an embodiment of the present invention.

detailed description

The specific implementation of the present invention will be further elaborated in conjunction with the accompanying drawings in the following description. The embodiments here are only used to illustrate the present invention, but are not used to limit the scope of the present invention.

As shown in Figure 1, it is a schematic diagram of the principle of the LED array light source microscopic imaging system. Based on the quantitative phase reduction microscopic imaging system provided by the present invention, the intensity I( _rc ) of the coherent point light source in the camera is:

I(r _c )＝|∫∫[∫∫q(r)o(r)exp(-i2πr·u″)d ² r]P(u″)exp(-i2πu″·r _c )d ² u″ | ² (1)

Among them, the illumination function of the LED array light source is q(r), the transmission function of the sample to be tested is o(r), the transformation function of other optical devices is P(u″), r _c is the coordinate of the focal plane of the camera, and u is the light The coordinates of the pupil plane;

When the sample is illuminated by an extended incoherent light source, then the light intensity distribution on the focal plane of the camera should be an incoherent superimposed image of a differential point light source. The intensity of the incoherent superimposed image of the above differential point light source is as follows:

I(r _c )＝∫∫|∫∫[∫∫q(r)o(r)exp(-i2πr·u″)d ² r]P(u″)exp(-i2πu″·r _c )d ² u″| ² d ² u′(2)

Since the LED array is far enough away from the sample carried by the microscope as the light source in the present invention, each lighting unit in the lighting pattern formed by the LED array is similar to a plane wave, then the lighting function of the LED array light source can be expressed as shown in the following formula:

Wherein, S(u') is the light intensity distribution function of the LED array light source illumination pattern, and u' is the coordinate on the plane of the LED array light source;

sample transmission function It is common sense in the field, μ(r) is the intensity transmittance, φ(r) is the phase change rate, approximately equivalent to o(r)≈1-μ(r)+iφ(r), according to the weak object approximation method Simplify the problem linearly, ignoring the cross term, the following expression can be obtained:

Substitute formula (3) and formula (4) into formula (2), and perform Fourier transform on both sides of the equation to obtain the light intensity frequency domain function Can be expressed by the following formula:

Formula (5) contains three aspects of information: background, absorption contrast and phase contrast:

Among them, the background term Bδ(u) in formula (5) is the sum of all the energy passing through the imaging system, which can be expressed as follows:

B＝∫∫S(u′)|P(u′)| ² d ² u′ (6)

The light intensity transfer function H _abs (u) in formula (5) can be expressed as follows:

H _abs (u)＝-[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′+∫∫S(u′)P ^* (u′)p(u′ -u)d ² u′] (7)

The phase transfer function H _ph (u) in formula (5) can be expressed as follows:

H _ph (u)=i[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′-∫∫S(u′)P ^* (u′)p(u′ -u)d ² u′] (8)

Based on the above derivation process, the present invention is based on establishing an equivalent relationship between differential phase contrast images and quantitative phase information. In order to derive the specific transfer function of differential phase contrast imaging, it is necessary to use two images of asymmetric illumination to calculate the differential phase contrast image; The flow chart of the imaging system restoring the quantitative phase image, the specific operation is as follows:

1. Build a microscope system based on LED array light source illumination;

2. LED array light source position correction;

3. The LED array light source forms an asymmetric lighting pattern design;

4. The differential phase contrast image restores the quantitative phase image;

Describe in detail below in conjunction with accompanying drawing and embodiment of description:

Step 1: Build a quantitative phase reduction microscopic imaging system, including a transmission microscope, the light source of the transmission microscope is an LED array, the illumination pattern of the LED array is controlled by programming, and it also includes multi-dimensional displacement for correcting the position of the LED array light source Platform, photographic equipment for collecting microscope observations and processing equipment for restoring the collected images to quantitative phase images.

Further, during the construction process, the camera is placed at the front end of the microscope system, and a high-speed camera with a single-mode frame rate of ≧24 Hz is used (as shown in Figure 3 is a schematic structural diagram of the microscopic imaging system of the present invention) and a controller is used to connect the LED array light source and the camera, so as to realize the multi-mode switching of the lighting pattern of the LED array light source and the synchronization of the camera acquisition operation in different modes.

In a preferred embodiment, the LED array is placed on a multi-dimensional displacement platform, and the multi-dimensional displacement platform is placed on the microscope object platform by 60-70 mm. The LED array light source can adopt any array size such as 32×32, 16×16, 8×8, 4×4, etc. that satisfies the NA value of the numerical aperture of the objective lens.

The asymmetric lighting pattern of the present invention is a semicircle or a semicircle with the center of the LED array as the center, or a rectangle symmetrical to the center of the LED array. The lighting pattern is a first lighting pattern and a second lighting pattern. The first lighting pattern and the The second lighting pattern is symmetrical about the center of the LED array, and the first lighting pattern and the second lighting pattern are simultaneously lit to form a circle, a ring or a rectangle with the center of the LED array as a midpoint.

The following further illustrates the relationship between the selection of the size of the LED array and the numerical aperture of the objective lens in the implementation manner that the illumination pattern is a semicircle with the center of the LED array as the center:

The numerical aperture expression of the microscope objective lens is as follows:

Wherein, n is the medium refractive index, and n=1 in the concrete example among the present invention; θ is the maximum angle that enters objective lens light; R is that the emitted light of LED array light source can enter the maximum radius of microscope objective lens, and D is that LED array light source and microscope Objective lens spacing.

It can be seen from the following formula that the minimum radius R of the LED array light source forming the lighting pattern is as follows:

If the microscope objective lens with 10× and numerical aperture of 0.25 is selected, the actual calculation is as follows:

The distance between the centers of any two adjacent light-emitting diodes in the LED array light source used in the embodiment of the present invention is 4 mm, therefore, the selected array size should be at least 8×8.

After determining the bright field range of the LED array according to the selected objective lens, the position of the LED array light source needs to be corrected in order to achieve accurate imaging.

Step 2: Correct the position of the LED array light source; this step mainly includes aligning the center of the LED array light source with the center of the objective lens, calibrating the horizontality of the LED array light source, and calibrating the direction angle of the LED array light source; as shown in Figure 4, the LED array light source Basic operation of the position calibration process:

Step 10: It is necessary to select the appropriate type of eyepiece and objective lens. Since the center of the LED array needs to be aligned with the center of the phase ring of the objective lens, it is necessary to select a phase type objective lens as the microscope objective lens, and then rotate the eyepiece of the microscope to port B. In this embodiment Bertram lens is used, so that the Fourier surface can be seen, that is, the LED array surface;

Step 11: Light the distance from the center of the LED array; in this implementation, the center of the LED array light source is used as the center, and the radius is two light-emitting diodes to facilitate center alignment;

Step 12: Align the center of the LED array with the center of the phase ring of the objective lens by adjusting the multi-dimensional displacement platform;

Since the acquisition of the differential phase contrast image requires high precision in the horizontality and directionality of the LED array light source, the naked eye calibration cannot meet the required accuracy. As a preferred implementation, machine vision technology is used to complete the calibration:

Step 21: Build the camera on the side port of the microscope;

Step 22: Place a suitable lens between the camera and the side port of the microscope, and adjust its relative position to the camera. According to the Fourier change characteristics of the lens, the camera can image the Fourier surface (and the LED array surface);

Step 23: Measure the levelness and direction angle of the LED array through the PC-side camera software, and complete the levelness and calibration by adjusting the multi-dimensional displacement platform.

After the position correction of the LED array light source is completed, the bright field area needs to be further divided in order to realize the differential phase contrast imaging.

Step 3: Design the LED array light source to form an asymmetrical lighting pattern;

In a preferred embodiment, the lighting pattern formed by the LED array light source adopts a static screen instead of a scanning method, because the static screen lighting method can increase the amount of light passing through. According to the mechanism of differential phase contrast imaging, it is common knowledge in the field that an asymmetric illumination pattern is required to obtain a phase contrast image. In a preferred embodiment, a semicircular lighting pattern with the center of the LED array as the center is usually used. In addition, the asymmetric lighting pattern of the present invention can also be a semicircular shape with the center of the LED array as the center or a rectangle symmetrical to the center of the LED array. .

The differential phase contrast imaging is obtained with a single axis of symmetry as the division direction, the illumination patterns are the first illumination pattern and the second illumination pattern, and the first illumination pattern and the second illumination pattern are complementary, that is: the first illumination pattern 1 and the The second lighting pattern is symmetrical about the center of the LED array, and the first lighting pattern and the second lighting pattern are simultaneously lit to form a circle, a ring or a rectangle with the center of the LED array as a midpoint.

As shown in Figure 5, the lighting pattern in this implementation is a semicircle with the center of the LED array as the center, and is divided into area 1 and area 2 by a single symmetrical axis with an angle α with the horizontal direction of the paper, and the division direction is not limited As shown in the accompanying drawings of the specification, the value range of α is 0°～180°; Imaging results. In order to obtain the phase contrast imaging results in various directions, the phase contrast imaging results along the α+90° direction can be obtained by adopting the transformation division method along the angle α.

Step 4: processing of differential phase contrast image reduction quantitative phase image;

The controller is used to control the switching of the lighting pattern of the LED array light source and the frequency synchronization of the exposure time of the camera to complete the acquisition of the target image under the illumination of the asymmetrical pattern formed by the division of a single symmetry axis. The first target image collected under the first differential illumination light is defined as I ₁ , the second target image under the second differential illumination light is I ₂ , and then the processing equipment is used for calculation and processing, and the specific processing method is as follows:

Step A: Obtain the differential phase contrast imaging picture I _DPC divided by any symmetry axis direction of the sample; define the first target image collected under the first differential illumination as I ₁ , and define the second target image under the second differential illumination as I ₂ , then the method to obtain _IDPC is:

For an aberration-free system, the pupil function is a circular function (real symmetry) of the aperture with the radius set to the target value. In this case, the absorbing transfer function make Formula (5) can be simplified to the following expression, that is, the relationship between the Fourier spectrum of the differential phase contrast image and the quantitative phase system:

Therefore, yes The quantitative phase information of the microscopic test of the sample can be obtained by performing the inverse Fourier transform, that is, the quantitative phase information of the microscopic test of the sample can be obtained through the frequency domain function of the differential phase contrast image.

As shown in Figure 5, the differential phase contrast imaging result obtained by dividing a single symmetry axis with any included angle is the phase contrast imaging result of the sample in the direction of α+90°.

Since the above differential phase contrast imaging result is a differential phase contrast image acquired in the direction of a single axis of symmetry, it does not contain phase information perpendicular to the axis of symmetry; in order to enhance the ability of phase reconstruction in different directions, multi-axis imaging can be used along any direction Divide and form an asymmetric illumination pattern, so as to obtain differential phase contrast imaging pictures containing phase information in different directions, and then further complete the differential phase contrast imaging containing different phase information and restore them to quantitative phase pictures. Furthermore, the present invention adopts multi-directional symmetrical axis division to form an asymmetrical lighting pattern, and then uses a processing device to process target images containing different phase information. The specific processing method is as follows:

Step A: Obtain differential phase contrast imaging pictures divided by any two or more symmetry axis directions of the sample

Define that when the illumination pattern is divided by the first axis of symmetry, the first target image collected by the first differential illumination light is I ₁₁ , and the second target image collected by the second differential illumination light is I ₁₂ ; the definition is symmetric by the jth When the illumination pattern is further divided by the axis, the first target image collected by the first differential illumination light is I _j1 , and the second target image collected by the second differential illumination light is I _j2 ; then the differential phase contrast image is:

Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function, and its expression is as follows:

H(u)=i[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′-∫∫S(u′)P ^* (u′)p(u′- u)d ² u′]

Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane;

Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows:

Solving the above equations jointly, the quantitative phase information based on multi-axis division is calculated by the following formula:

Since the frequency point at the intersection of H(u) and the axial edge is zero and the frequency involved in the operation is wide, direct deconvolution will greatly superimpose the noise on these frequencies, so the problem can be solved by the regularized least squares method, and finally The simplified quantitative phase information expression is as follows: where, α is the regularization parameter

The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention, and does not limit the present invention. All modifications, equivalent replacements and improvements made within the application scope of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A method for restoring quantitative phase images based on differential phase contrast images, characterized in that, comprising the following steps: Step A: Using a program-controlled LED array as a light source, controlling the switching of the lighting pattern of the LED array light source based on the time-division multiplexing mode to synchronize with the frequency of image acquisition by the photographic equipment, wherein the target image is collected under an asymmetrical lighting pattern, and the asymmetrical The symmetrical illumination pattern is divided by a single axis of symmetry; define the first target image collected under the first differential illumination as I ₁ , and the second target image under the second differential illumination as I ₂ , then the differential phase contrast image is as follows : ${\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}\mathrm{<span\; class="notranslate">\; P</span>}\mathrm{<span\; class="notranslate">\; C</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}$ Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function, and its expression is as follows: H(u)=i[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′-∫∫S(u′)P ^* (u′)p(u′- u)d ² u′] Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane; Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows: Then the quantitative phase information restored in the Fourier domain is finally to Quantitative phase information is obtained by inverse Fourier transform. 2. A method for restoring quantitative phase images based on differential phase contrast images, characterized in that, comprising the following steps: Step A: Using a program-controlled LED array as a light source, controlling the switching of the lighting pattern of the LED array light source based on the time-division multiplexing mode to synchronize with the frequency of image acquisition by the photographic equipment, wherein the target image is collected under an asymmetrical lighting pattern, and the asymmetrical The symmetrical lighting pattern is divided by two or more symmetric axes in any direction; the acquisition of the target image is performed according to the following sequential steps: A1: Use the first axis of symmetry to divide the illumination pattern, define the first target image acquired under the first differential illumination as I ₁₁ , and define the second target image acquired under the second differential illumination as I ₁₂ ; A2: Use the jth symmetry axis to further divide the illumination pattern, define the first target image collected under the first differential illumination as I _j1 , and define the second target image collected under the second differential illumination as I _j2 ; A3: Repeat the operation of step A2 until the target axis number is reached, then the differential phase contrast images of the two target images obtained in each step are as follows: ${\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}\mathrm{<span\; class="notranslate">\; P</span>}\mathrm{<span\; class="notranslate">\; C</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; j</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}}$ Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function: H(u)=i[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′-∫∫s(u′)P ^* (u′)p(u′- u)d ² u′] Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane; Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows: Solving the above equations jointly, the quantitative phase information based on multi-axis division is calculated by the following formula: 3. A method for restoring quantitative phase images based on differential phase contrast images according to claim 1 or 2, wherein the LED array is a rectangular array formed by m×n light-emitting diodes arranged at equal intervals, m , n are both positive integers; the LED array lighting pattern is lit using a static screen mode. 4. A method for restoring a quantitative phase image based on a differential phase contrast image according to claim 1 or 2, wherein the asymmetric illumination pattern is a semicircle or a semicircle with the center of the LED array as the center, or is a The center of the LED array is a symmetrical rectangle, the lighting pattern is a first lighting pattern and a second lighting pattern, the first lighting pattern and the second lighting pattern are symmetrical about the center of the LED array, and the first lighting pattern and the second lighting pattern Simultaneous lighting forms a circle, ring or rectangle with the center of the LED array as the midpoint. 5. A method for restoring a quantitative phase image based on a differential phase contrast image according to claim 1 or 2, characterized in that, the position correction of the LED array light source is also included before the microscopic imaging system of the LED array light source is built for image acquisition , including aligning the center of the LED array with the center of the microscope objective lens, calibrating the horizontality of the LED array, and calibrating the azimuth of the LED array. 6. A quantitative phase reduction microscopic imaging system, characterized in that it comprises a transmission microscope, wherein the light source of the transmission microscope is an LED array, and the illumination pattern of the LED array is controlled by programming, and also includes a The multi-dimensional displacement platform for correcting the position of the LED array light source, the camera equipment for collecting the observation results of the microscope, and the processing equipment for restoring the collected images to quantitative phase images. 7. A quantitative phase reduction microscopic imaging system according to claim 6, wherein the LED array is a rectangular array composed of m×n light-emitting diodes arranged at equal intervals, and m and n are both positive integers ; The lighting pattern of LED array lighting adopts static screen mode. 8. A kind of quantitative phase reduction microscopic imaging system according to claim 6, wherein the processing device obtains the quantitative phase imaging picture of the sample according to the collected target image, and the specific method is: Step A: Obtain the differential phase contrast imaging picture I _DPC divided by any symmetry axis direction of the sample; define the first target image collected under the first differential illumination as I ₁ , and define the second target image under the second differential illumination as I ₂ , then the method to obtain _IDPC is: ${\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}\mathrm{<span\; class="notranslate">\; P</span>}\mathrm{<span\; class="notranslate">\; C</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}$ Step B: Establish the two-dimensional optical phase transfer function H(u) of differential phase contrast imaging based on the weak target transfer function, and its expression is as follows: H(u)=i[∫∫S(u′)P ^* (u′)p(u′+u)d ² u′-∫∫S(u′)P ^* (u′)p(u′- u)d ² u′] Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane; Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows: Then the quantitative phase information restored in the Fourier domain is right The inverse Fourier transform is performed, and quantitative phase information is finally obtained. 9. A kind of quantitative phase reduction microscopic imaging system according to claim 6, characterized in that, the processing device obtains the quantitative phase imaging picture according to the collected target image, and its specific method is: Step A: Obtain differential phase contrast imaging pictures divided by any two or more symmetry axis directions of the sample Define that when the illumination pattern is divided by the first axis of symmetry, the first target image collected by the first differential illumination light is I ₁₁ , and the second target image collected by the second differential illumination light is I ₁₂ ; the definition is symmetric by the jth When the illumination pattern is further divided by the axis, the first target image collected by the first differential illumination light is I _j1 , and the second target image collected by the second differential illumination light is I _j2 ; then the differential phase contrast image is: ${\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}\mathrm{<span\; class="notranslate">\; P</span>}\mathrm{<span\; class="notranslate">\; C</span>}<span\; class="notranslate">\; ,</span>\mathrm{<span\; class="notranslate">\; j</span>}}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; j</span>}\mathrm{<span\; class="notranslate">\; 2</span>}}}$ Step B: Establish a two-dimensional optical phase transfer function H(u) for differential phase contrast imaging based on the weak target transfer function, and its expression is as follows: H(u)=i[∫∫s(u′)P ^* (u′)p(u′+u)d ² u′-∫∫s(u′)P ^* (u′)p(u′- u)d ² u′] Among them, S(u') is the light intensity distribution function of the light source, P(u') is the pupil transfer function, and u' is the coordinate on the pupil plane; Step C: Establish the relationship model between the differential phase contrast image and the phase information in the Fourier domain as follows: Solving the above equations together, the quantitative phase information based on multi-axis division is calculated by the following formula: 10. A quantitative phase reduction microscopic imaging system according to any one of claims 6 to 9, further comprising a controller, the controller is respectively connected with the LED array light source and the photographic equipment to control the LED array The multi-mode lighting pattern switching is synchronized with the frequency of image acquisition by the camera equipment.