CN109780993A - Differential Phase Contrast Microscopy System and Method - Google Patents

Abstract

A differential phase contrast microscopy system and method. The system includes a light intensity modulation module, a condenser lens, an objective lens and an image capture module. The light intensity modulation module is used to modulate the incident light field into a detection light field with a gradient intensity distribution according to the control signal. The condenser lens is used to receive the detection light field, generate an off-axis light field and project it onto an object to be measured, and then generate a light field of the object to be measured which is received by the objective lens. The image capture module is used to receive the light field of the object to be measured, thereby generating an optical image corresponding to the intensity gradient distribution. The method further includes controlling the light intensity modulation module to generate a first gradient shielding in which the light intensity gradually decreases along a first axis, a second gradient shielding in which the light intensity gradually increases along the first axis, and a second gradient shielding in which the light intensity gradually increases along a second axis. The gradually decreasing third gradient shielding and the gradually increasing fourth gradient shielding with light intensity along the second axis enable the image capture module to respectively capture first to fourth optical images corresponding to the first to fourth gradient shielding.

Description

Differential Phase Contrast Microscopy System and Method

technical field

The invention relates to a microscopic technique, in particular to a differential method that utilizes a biaxial light field with light intensity gradient distribution to obtain four corresponding images about an object to be measured by off-axis illumination, so as to perform phase measurement. Phase contrast microscopy systems and methods.

Background technique

Differential phase contrast microscopy (DPC) is a technology that uses non-interference images to analyze the characteristics of objects. phase contrast under conditions. Due to its label-free advantage, differential phase-contrast microscopy has been widely used in commercial assays to observe biological specimens without photobleaching or phototoxicity.

In the field of non-visible light in the conventional technology, there is a differential microscopy technique that uses an X-ray source and a grating to generate phase contrast. For example, Chinese Application Publication No. CN103348415 discloses a method of the present invention involving X-ray differential phase contrast imaging. To enhance the information acquired by phase contrast imaging, analysis gratings for X-ray differential phase contrast imaging are provided with absorbing structures. The latter includes a first plurality of first regions with a first X-ray attenuation and a second plurality of second regions with a second X-ray attenuation. The second X-ray attenuation is smaller than the first X-ray attenuation, and the first and second regions are periodically arranged in an alternating manner. A third plurality of third regions are provided with a third X-ray attenuation within a range from said second X-ray attenuation to said first X-ray attenuation, wherein every second said first region Or every second of said second regions is replaced by one of said third regions.

In addition, in a typical DPC architecture using visible light sources for detection, the light source is modulated using a semi-circular pattern, which, as shown in Figure 1A, can be modulated using a light intensity mask or a programmable LED array. Another way is to use a spatial light modulator (SLM) or a liquid crystal panel arranged on the Fourier plane (Fourier plane) of the objective lens to generate detection light. Spectral modulation of light intensity shielding for semicircular patterns utilizes the Hilbert transform, a technique that has been demonstrated to be capable of coherent laser illumination and placement of spatial light modulation modules on the Fourier plane of the objective. A phase with an isotropic phase contrast response is obtained. However, since under partially coherent illumination, the DPC transfer function is anisotropic when measured with only two-axis (vertical and horizontal) semicircular light intensity masks, it is necessary to To perform up to twelve axially varying light intensity shields, as shown in Figure 1B, the off-axis phase varying shields 00-11 are measured to improve the stability and accuracy in the phase restoration calculation. In addition, since the use of the semicircular light intensity shield requires up to twelve axial light intensity changes, the measurement efficiency is also greatly reduced, which is not conducive to the detection on the production line.

To sum up the above, in order to solve the problems existing in the DPC of the visible light source, the present invention needs a differential phase contrast microscope system and method to solve the problems in the prior art.

SUMMARY OF THE INVENTION

The invention provides a differential phase contrast microscope system and method, which utilizes the shielding of four different light intensity gradient distributions on two axes, modulates incident light and performs off-axis illumination on the object to be measured, and the formed object light passes through the objective lens Four images are captured by the image capture device. Due to the local coherent illumination of the present invention, only four different light intensity gradient distributions are used for shielding, that is, the phase of each detection position on the object to be tested can be restored by capturing four correspondingly shielded images, and then the surface of the object can be known. Therefore, the invention has the effects of saving the time required for measurement, reducing the noise of the coherent light spot, and enhancing the resolution.

In one embodiment, the present invention provides a differential phase contrast microscope system, which includes a light source, a light intensity modulation module, a condenser lens, an objective lens, and an image capture module. The light source is used to generate an incident light field. The light intensity modulation module is used for generating, according to the control signal, a first gradient shield whose light intensity gradually decreases along a first axis, a second gradient shield whose light intensity gradually increases along the first axis, and a second gradient shield along a second axis One of a third gradient mask whose light intensity gradually decreases and a fourth gradient mask whose light intensity gradually increases along the second axis is used to modulate the incident light field into a detection light field with an intensity gradient distribution. The condenser lens is arranged on one side of the light intensity modulation module, so that the light intensity modulation module is located on the Fourier plane of the condenser lens, and the condenser lens is used to receive the detection light field and generate an off-axis The light field is projected onto an object to be measured, thereby generating an object light field. The objective lens is arranged on one side of the condensing lens, so that the object to be side is located on the focal length of the objective lens, and the objective lens receives the light field of the measuring object. The image capturing module is coupled to the objective lens for receiving the object light field, thereby generating an optical image corresponding to the intensity gradient distribution.

In one embodiment, the light intensity modulation module is a liquid crystal light transmission module, which has a liquid crystal unit for changing the light transmission amount according to a control signal. In addition, in another embodiment, the condenser lens has a first numerical aperture value, the objective lens has a second numerical aperture value, and the first and second numerical aperture values are 1.

In one embodiment, the present invention provides a differential phase contrast microscopic image capturing method, which includes the following steps. First, a light source generates an incident light field. Next, a light intensity modulation module is provided for modulating the incident light field into a detection light field with an intensity gradient distribution according to the control signal. Then, a condenser lens is provided, which is arranged on one side of the light intensity modulation module, so that the light intensity modulation module is located on the Fourier plane of the condenser lens, and the condenser lens is used to receive the detection light field and generate An off-axis light field is projected onto an object to be measured, thereby generating an object light field. Next, an objective lens is provided, which is arranged on one side of the condensing lens, so that the object to be side is located on the focal length of the objective lens, and the objective lens receives the light field of the measuring object. Next, an image capturing module is provided coupled to the objective lens for generating an optical image corresponding to the detected light field. Next, the light intensity modulation module is controlled to generate a first gradient shield whose light intensity gradually decreases along a first axis, a second gradient shield whose light intensity gradually increases along the first axis, and a light intensity along a second axis A third gradient mask that gradually decreases and a fourth gradient mask that gradually increases light intensity along the second axis. Finally, make the image capture module capture the first optical image corresponding to the first gradient mask, the second optical image corresponding to the second gradient mask, the third optical image corresponding to the third gradient mask, and the A fourth optical image that should be a fourth gradient mask.

In another embodiment, the differential phase contrast microscopic image capturing method further includes using an arithmetic processing unit to receive the first optical image, the second optical image, the third optical image and the fourth optical image , and perform calculation to obtain the phase of each detection position on the test object, and then reconstruct the surface topography of the test object. Wherein, performing calculation to obtain the phase of each detection position on the object to be tested further includes the following steps: (a) calculating the first phase from the light intensity corresponding to each detection position on the first and second optical images Compare the image IDPC and perform Fourier operation to obtain a converted first phase contrast image value; (b) calculate the second phase contrast image IDPC from the light intensity corresponding to each detection position on the third and fourth optical images and perform Fourier operation to obtain a transformed second phase contrast image value; (c) calculating the inner product of the transformed first phase contrast image value and a first transformation function and the inner product of the transformed second phase contrast image value and a second transformation function (d) adding the sum of the squares of the first transfer function and the second transfer function to a noise suppression function; (e) dividing the sum of step (c) by the addition of step (d) and (f) performing an inverse Fourier transform on the result of step (e) to obtain the phase corresponding to each detected position. The noise suppression function further includes a high frequency suppression function and a low frequency suppression function.

Description of drawings

FIG. 1A and FIG. 1B are schematic diagrams of the conventional light shield and the phase change with different axes;

2 is a schematic diagram of the optical structure of the differential phase contrast microscope system of the present invention;

3A-3D are schematic diagrams of shielding of changes in the light intensity gradient distribution along the first axis and the second axis according to the present invention;

4 is a schematic flowchart of the differential phase contrast microscopy method of the present invention;

5A to 5D are schematic diagrams of images captured using the mask of the present invention;

6A is a simulation diagram of a phase transfer function formed by a biaxial semicircular shield used in an existing differential phase contrast microscope system;

6B is a simulation diagram of a phase transfer function formed by a biaxial shield with gradient distribution used by the differential phase contrast microscope system of the present invention;

Fig. 6C is the result of the intensity subtraction of the phase transfer function of the prior art and the present invention;

7A-7C are comparison diagrams between the present invention and the prior art after the cell image is captured and calculated;

FIG. 8A is a phase image reproduced by using the biaxial four images of the present invention;

8B is a reconstructed phase image using a 12-axis image measured by conventional DPC;

FIG. 8C is a phase segment graph along the solid line in FIG. 8A .

Description of reference numerals: 00-11-semicircle shield; 2-differential phase contrast microscope; 20-light source; 200-incident light field; 21-light intensity modulation module; 210-detection light field; 22-condenser lens ;220-off-axis light field;221-object light field;23-objective lens;24-image capture module;25-tubular lens;26-operation processing unit;3-differential phase contrast microscopy;30～37- Steps; 90 - object to be tested; 91 - first gradient shield; 92 - second gradient shield; 93 - third gradient shield; 94 - fourth gradient shield.

Detailed ways

Various illustrative embodiments may be described more fully hereinafter with reference to the accompanying drawings, in which some illustrative embodiments are shown. The inventive concepts may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these illustrative embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventive concept to those skilled in the art. Similar numbers always indicate similar components. The differential phase contrast microscope system and method will be described below with various embodiments in conjunction with the drawings, however, the following embodiments are not intended to limit the present invention.

Please refer to FIG. 2 , which is a schematic diagram of the optical structure of the differential phase contrast microscope system of the present invention. In this embodiment, the microscope system 2 includes a light source 20 , a light intensity modulation module 21 , and a condenser lens 22 . An objective lens 23 , an image capturing module 24 and an arithmetic processing unit 26 .

The light source 20 is used to generate an incident light field 200. In this embodiment, the light source is a broadband visible light source, but not limited thereto, for example, a monochromatic visible light source can also be implemented. The light intensity modulation module 21 is disposed on one side of the light source 20 , and is a module that generates a light intensity gradient distribution that can modulate the incident light field 200 according to a control signal. In one embodiment, the light intensity modulation module 21 is a liquid crystal module (TFT shield) capable of controlling the light penetration intensity or a liquid crystal on silicon (LCoS) module. These modules feature the ability to control the steering of the liquid crystal, which in turn changes the intensity of transmitted or reflected light. In the present invention, the light intensity modulation module 21 can generate light intensity gradient distribution shields along different axes according to the control signal, so as to modulate the incident light field 200 into a detection light field 210 with an intensity gradient distribution, for example : As shown in FIGS. 3A to 3D , in which FIG. 3A is a first gradient mask whose light intensity gradually decreases along a first axis, FIG. 3B is a second gradient mask whose light intensity gradually increases along the first axis, and FIG. 3C is a A third gradient mask with gradually decreasing light intensity along a second axis and a fourth gradient mask with gradually increasing light intensity along the second axis in FIG. 3D . In this embodiment, the first axis is the X axis, and the second axis is the Y axis, but this is not a limitation. The problem of the intensity cross produced in the middle-axis when measuring each axis by using a semicircular light-dark mask in the prior art can be solved by a mask with a light intensity gradient distribution.

The condenser 22 is disposed on one side of the light intensity modulation module 21 to receive the detection light field 210 modulated by the light modulation module 21 . The light intensity modulation module 21 is located on the Fourier plane of the condenser lens 22. The condenser lens 22 is used to receive the detection light field 210 and generate an off-axis light field 220 to project to an object to be measured. on the object 90, thereby generating an object light field 221. The objective lens 23 is disposed on one side of the condensing lens 22 , so that the object to be side 90 is located at the focal length of the objective lens 23 , and the objective lens 23 receives the object light field 221 that penetrates the object to be measured 90 . It should be noted that the structure of the present invention can generate an optical image of partially coherent illumination (partially coherent illumination). In one embodiment, the condition for generating partially coherent illumination is that the condenser lens 22 has a first numerical aperture. NA) value, the objective lens has a second numerical aperture value, and the first and the second numerical aperture values are 1 or approximately 1. Utilizing partial coherent illumination can produce effects superior to prior art coherent illuminations, such as resolution enhancement, increased optical sectioning effects, and reduced coherent speckle noise.

The image capturing module 24 is coupled to the objective lens 23 for receiving the object light field 221 to generate an optical image corresponding to the intensity gradient distribution. The distance between the image plane of the image capturing module 24 and the object 90 for capturing images is twice the focal length of the objective lens 23 and twice the focal length of the lens 25 . In this embodiment, the image capturing module 24 is coupled to the objective lens 23 through a tube lens 25 . The objective lens 23 and the tubular lens 25 in this embodiment are the structures of a microscope system, and the microscope system can be a commercial microscope system, such as Leica, DMI3000 equipment, but not limited thereto.

The arithmetic processing unit 26 is electrically connected to the light intensity modulation module 21 for generating a control signal to control the light intensity modulation module 21 to generate shielding along different axial light intensity gradients, for example, as shown in FIGS. 3A to 3D . a first gradient shield with gradually decreasing light intensity along a first axis, a second gradient shield with gradually increasing light intensity along the first axis, a third gradient shield with gradually decreasing light intensity along a second axis, and a second gradient shield with gradually decreasing light intensity along the first axis The second axially increasing fourth gradient of light intensity shields one of them. In addition, the arithmetic processing unit 26 is further electrically connected to the image capturing module 24 for receiving information about the first gradient mask 91 , the second gradient mask 92 , the third gradient mask 92 and the third gradient mask 91 captured by the image capturing module 24 . The first optical image, the second optical image, the third optical image and the fourth optical image of the gradient mask 93 and the fourth gradient mask 94 are calculated to obtain the phase of each detection position on the object to be tested, and then Reconstruct the surface topography or internal structural features of the test object. The arithmetic processing unit 26 is a computer, server or workstation with arithmetic processing capability, which can execute application programs from the storage medium and perform operations on the captured images.

Please refer to FIG. 2 and FIG. 4 , in which FIG. 4 is a schematic flowchart of the differential phase contrast microscopy method 3 of the present invention. In this embodiment, the method 3 uses the microscope system of FIG. 2 to perform differential phase on the object surface Contrast microscopic measurements. Step 30 is first performed to provide a differential phase contrast microscope system as shown in FIG. 2 . Next, step 31 is performed to enable the light source 20 to generate an incident light field 200 and project it to the light intensity modulation module 21 . Next, step 32 is performed to control the light modulation module 21 to modulate the incident light field 200 into a detection light field 210 having an intensity gradient distribution according to the control signal. In this step, firstly, the arithmetic processing unit 26 generates a control signal to control the light intensity modulation module 21 to first generate a first gradient mask 91 whose light intensity gradually decreases along a first axis, as shown in FIG. 3A . Therefore, when the incident light field passes through the first gradient shield 91, the incident light output will be modulated into a detection light field 210 whose light front is gradually reduced in intensity along the first axis.

Next, step 33 is performed to make the incident light field 210 pass through the condenser lens 22 disposed on one side of the light intensity modulation module 21 . The light intensity modulation module 21 is located on the Fourier plane of the condenser lens 22. The condenser lens 22 is used to receive the detection light field 210 and generate an off-axis light field 220 to project on an object to be measured 90, and then An object light field 221 is generated. The analyte may be a cell or a microstructure, and this embodiment is a cell. Next, step 34 is performed, so that the object light field 221 is received by the objective lens 23 , and the object to be sided 90 is located at the focal length of the objective lens 23 . Next, step 35 is performed to provide an image capture module coupled to the objective lens for generating a first optical image corresponding to the detection light field of the first gradient mask whose light intensity gradually decreases along the first axis, as shown in FIG. 5A . Show.

Next, step 36 is performed to determine whether four images corresponding to different axial light intensity gradient distributions have been acquired. If not, step 37 is performed to change the light intensity gradient of the light intensity modulation module 21 . In this step, the arithmetic processing unit 26 controls the light intensity modulation module 21 to generate a second gradient mask whose light intensity gradually increases along a first axis. Then go back to step 33, and repeat steps to 35 to obtain a second optical image corresponding to the detection light field of the second gradient mask with the light intensity gradually increasing along the first axis, as shown in FIG. 5B. After that, step 37 is performed to change the light intensity gradient of the light intensity modulation module 21 . The arithmetic processing unit 26 is made to control the light intensity modulation module 21 to generate a third gradient mask whose light intensity gradually decreases along a second axis. Then go back to step 33, and repeat steps to 35 to obtain a third optical image corresponding to the detection light field of the third gradient mask whose light intensity gradually decreases along the second axis, as shown in FIG. 5C. After that, step 37 is performed to change the light intensity gradient of the light intensity modulation module 21 . The arithmetic processing unit 26 controls the light intensity modulation module 21 to generate a fourth gradient mask whose light intensity gradually increases along a second axis. Then go back to step 33, and repeat steps to 35 to obtain a fourth optical image corresponding to the detection light field of the fourth gradient mask with the light intensity gradually increasing along the second axis, as shown in FIG. 5D.

After the four optical images corresponding to the gradual increase and decrease of the gradients in the two axes are obtained, step 38 is then performed by the arithmetic processing unit 26 to perform arithmetic processing and analysis, so as to analyze the received first optical image, the second optical image, The third optical image and the fourth optical image are calculated to obtain the phase of each detection position on the object to be tested 90, and then the topography or internal features of the object to be tested are reconstructed.

In one embodiment, taking FIG. 5A to FIG. 5D as an example, the calculation to obtain the phase of each detection position on the object to be tested further includes the following steps: firstly, step 370 is performed, and the first and second optical images are obtained from the first and second optical images. The first phase contrast image IDPC is calculated from the light intensity corresponding to each detection position and Fourier calculation is performed to obtain a converted first phase contrast image value. Where i=1, represents the first axis X, r(x, y) represents each detection position (x, y), and I _DPC is shown in the following formula (1).

I _DPC = (I ₁ -I ₂ )/(I ₁ +I ₂ ).....(1)

In this step, I ₁ in the formula (1) is the light intensity value corresponding to each detection position in the first image, and I ₂ is the light intensity value corresponding to each detection position in the second image.

Then go to step 371, calculate the second phase contrast image IDPC from the light intensity corresponding to each detection position on the third and fourth optical images and perform Fourier operation to obtain a converted second phase contrast image value Where i=2, represents the first axis Y, r(x, y) represents each detection position (x, y), and the I _DPC value is calculated by the above formula (1). In this step, I ₁ in the formula (1) is the light intensity value corresponding to each detection position in the third image, and I ₂ is the light intensity value corresponding to each detection position in the fourth image.

Then proceed to step 372 to calculate the converted first phase contrast image value with a first conversion function The inner product and the transformed second phase contrast image value with a second conversion function the sum of inner products, where As shown in Equation (2) below, the summation of step 372 is shown in Equation (3) below.

where H _p,1 (u) and H _p,2 (u) are the image pairs on each axis, respectively, is the background of the light field, such as the phase transfer function (pTF) of the corresponding masks on S(u) of the first and second images, or the third and fourth images, and S(u) is defined as the following formula (4):

S(u)=m(u)circ(u/ρ _c )....(4)

Wherein, u=(ux, uy) is defined as spatial frequency coordinates, and m(u) is the corresponding function of the shielding generated by the light intensity modulation module 21 . ρ _c =NA _condenser /λ, where NA _condenser represents the numerical aperture value of the condenser lens 22 , λ is the operating wavelength of the incident light field, and circ(ξ) is defined as the following equation (5):

Please refer to FIGS. 6A to 6C , wherein FIG. 6A is a simulation diagram of a phase transfer function formed by a biaxial semicircular shield used in a conventional differential phase contrast microscope; FIG. 6B is a differential phase contrast of the present invention. The simulation diagram of the phase transfer function formed by the biaxial shield with gradient distribution used in the microscope system; FIG. 6C is the result of the subtraction of the intensities of the phase transfer function of the prior art and the present invention. In FIG. 6A , the first graph represents the phase transfer function image of the horizontal axis, the second graph represents the phase transfer function image of the vertical axis, and the third graph represents the phase transfer function image of the two axes, and in FIG. 6B , the first graph The phase transfer function image represents the horizontal axis, the second graph represents the vertical axis phase transfer function image, and the third graph represents the dual axis phase transfer function image. It can be seen from the biaxial phase transfer function images in FIG. 6A and FIG. 6B , in FIG. 6A , the intensity of the transfer function shows an inhomogeneous result, while the intensity of the transfer function presented in the present invention shows a local Under the coherent illumination, there is an isotropic uniform distribution of the transfer function image like a donut. Therefore, the present invention can reduce the noise of the coherent light spot and enhance the resolution and other effects by using the transfer function image generated by the mask with the intensity gradient change. .

Next, step 373 is performed, and the square sum of the first transfer function and the second transfer function is added to a noise suppression function to form the result of the following formula (6).

in, represents the noise suppression function, which further includes a high frequency suppression function are the first-order differential operators along the vertical and horizontal axes, and a low-frequency suppression function in, represents the scaling function to suppress low-frequency noise, and _σw is the standard deviation. η, α, and β are adjustment parameters, and in one embodiment, they are 1, 10-2 to 10-3, and 10-3 to 10-4, respectively.

Step 374 is then performed to divide the sum of step (c) by the value obtained from the addition of step (d). And finally, step 375 is performed, as shown in the following formula (7), inverse Fourier transform is performed on the result of step (e) to obtain the phase corresponding to each detection position.

After obtaining the phase of each detection position, the characteristics of the object, such as surface topography, can be restored according to the phase value.

Furthermore, Equation (7) is derived from Equation (8) below.

in, represents the scaling function to suppress low-frequency noise, and _σw is the standard deviation. also, It is an adjustment item used to prevent noise from being amplified in the high frequency region. To solve Equation (8), let Lagrangian be 0 through differentiation, that is, the phase value equation of Equation (7) above can be derived.

Different from the traditional analytical method shown in Equation (9) below, in Equation (9), represents the transfer function, and I _DPC,i (r) represents the phase contrast image of each axis, Represents the Fourier transform calculus.

In the present invention, in the existing phase reduction equation, the constant γ is further adjusted as The resolution of phase restoration is improved by processing high and low frequency noise on the pair of biaxial images (the first and second images and the third and fourth images) obtained by the present invention.

Please refer to FIG. 7A to FIG. 7C , which are comparison diagrams of the present invention and the prior art after the cell image is captured and calculated. 7A is the phase image image restored by using equation (7), FIG. 7B is the phase image image restored by using equation (9), and FIG. 7C is the box area in FIG. 7A and the corresponding area in FIG. 7B Schematic diagram of the phase difference of the region. It can be seen from Figure 7C that the phase difference is close to 0 and shows a ringing artifacts pattern due to noise. Using the phase restored by equation (7), the noise can be effectively removed and the image quality can be improved.

Next, the measurement of an object with a semicircular surface structure using the method of the present invention and a conventional DPC will be described. Please refer to FIG. 8A and FIG. 8B , wherein FIG. 8A is a phase image reconstructed by using the dual-axis four images of the present invention, and FIG. 8B is a reconstructed phase image using a 12-axis image measured by a conventional DPC. In the lower left corner of FIG. 8A and FIG. 8B , the phase function simulated by multi-axis is used in detail. It can be seen from FIG. 8A , that is, using the method of the present invention, the phase function is the structure of a doughnut with good isotropy, and The lower left corner of Figure 8B shows the phase function of the 12-axis image measured by the traditional DPC. It can be seen that it has a shape similar to a rectangle, so the reconstructed phase image is anisotropic, with only a cross in the middle. The regions are relatively accurate, far from the isotropic phase images presented in Figure 8A. As shown in FIG. 8C , the graph is a phase segment graph along the solid line in FIG. 8A . The solid line is the measured phase value, while the dashed line is the predicted value calculated using data provided by the lens array manufacturer. It can be seen that the actual measurements are quite close to the predicted values using mathematical calculations.

The above description merely describes the preferred embodiments or embodiments of the present invention for presenting the technical means used to solve the problem, and is not intended to limit the scope of the patent implementation of the present invention. That is, all the equivalent changes and modifications that are consistent with the meaning of the claims of the present invention, or made according to the claims of the present invention, are all covered by the scope of the patent of the present invention.

Claims (10)

1. a differential phase contrast microscope system, is characterized in that, comprises: a light source for generating an incident light field; a light intensity modulation module for generating, according to the control signal, a first gradient mask whose light intensity gradually decreases along a first axis, a second gradient mask whose light intensity gradually increases along the first axis, and a second gradient mask along a second axis one of a third gradient shield whose light intensity gradually decreases and a fourth gradient shield whose light intensity gradually increases along the second axis, so as to modulate the incident light field into a detection light field with an intensity gradient distribution; a condenser lens, disposed on one side of the light intensity modulation module, so that the light intensity modulation module is located on the Fourier plane of the condenser lens, the condenser lens is used to receive the detection light field and generate an off-axis The light field is projected onto an object to be measured, thereby generating a light field of the object to be measured; an objective lens, arranged on one side of the condenser lens, so that the object to be side is located at the focal length of the objective lens, and the objective lens receives the object light field; and An image capturing module is coupled to the objective lens for receiving the object light field to generate an optical image corresponding to the intensity gradient distribution. 2 . The differential phase contrast microscope system according to claim 1 , wherein the light intensity modulation module is a liquid crystal module for controlling light penetration intensity or a light reflection type liquid crystal module, and the light intensity modulation module has a liquid crystal module inside. 3 . unit, the liquid crystal unit is used to change the light transmission amount according to the control signal. 3. The differential phase contrast microscope system of claim 1, wherein the condenser lens has a first numerical aperture value, the objective lens has a second numerical aperture value, and the first numerical aperture value is the same as the The second numerical aperture values are all 1. 4 . The differential phase contrast microscope system of claim 1 , further comprising an arithmetic processing unit for generating a control signal to enable the light intensity modulation module to select the light intensity gradually decreasing along a first axis. 5 . a first gradient shield, a second gradient shield with gradually increasing light intensity along the first axis, a third gradient shield with gradually decreasing light intensity along a second axis, and a fourth gradient shield with gradually increasing light intensity along the second axis one of the gradient masks, the arithmetic processing unit further receives the first optical image, the second optical image, the third optical image about the first gradient mask, the second gradient mask, the third gradient mask and the fourth gradient mask The image and the fourth optical image are used for calculation to obtain the phase of each detection position on the object to be tested, so as to reconstruct the surface topography of the object to be tested. 5. A differential phase contrast microscopic image capturing method, characterized in that, comprising the following steps: causing a light source to generate an incident light field; providing a light intensity modulation module for modulating the incident light field into a detection light field with an intensity gradient distribution according to the control signal; A condenser lens is provided, which is arranged on one side of the light intensity modulation module, so that the light intensity modulation module is located on the Fourier plane of the condenser lens, and the condenser lens is used to receive the detection light field and generate a separation The axial light field is projected onto an object to be measured, thereby generating an object light field; Provide an objective lens, which is arranged on one side of the condenser lens, so that the object to be side is located on the focal length of the objective lens, and the objective lens receives the light field of the measuring object; providing an image capture module coupled to the objective lens for generating an optical image corresponding to the detected light field; The light intensity modulation module is controlled to generate a first gradient shield with a gradually decreasing light intensity along a first axis, a second gradient shield with a gradually increasing light intensity along the first axis, and a light intensity along a second axis a third gradient mask that gradually decreases and a fourth gradient mask that gradually increases light intensity along the second axis; and causing the image capture module to capture a first optical image corresponding to the first gradient mask, a second optical image corresponding to the second gradient mask, a third optical image corresponding to the third gradient mask, and a third optical image corresponding to the third gradient mask through the objective lens Fourth optical image of the four gradient mask. 6 . The method of claim 5 , wherein the light intensity modulation module is a liquid crystal module for controlling light penetration intensity or a light reflection type liquid crystal module, the light intensity modulation module is 6 . There is a liquid crystal unit inside, and the liquid crystal unit is used to change the amount of light transmission according to the control signal. 7. The method of claim 5, wherein the condenser lens has a first numerical aperture value, the objective lens has a second numerical aperture value, and the first numerical aperture value value and the second numerical aperture value are both 1. 8. The differential phase contrast microscopic image capturing method of claim 5, further comprising the following steps: An arithmetic processing unit is used to receive the first optical image, the second optical image, the third optical image and the fourth optical image, and perform calculation to obtain the phase of each detection position on the object to be tested, and then reconstruct The surface morphology of the test object. 9. The differential phase contrast microscopic image capturing method as claimed in claim 8, wherein the calculation to obtain the phase of each detection position on the object to be measured further comprises the following steps: (a) calculating the first phase contrast image IDPC from the light intensity corresponding to each detection position on the first and second optical images and performing Fourier operation to obtain a converted first phase contrast image value; (b) calculating the second phase contrast image IDPC from the light intensity corresponding to each detection position on the third and fourth optical images and performing Fourier operation to obtain a converted second phase contrast image value; (c) calculating the sum of the inner product of the converted first phase-contrast image value and a first transfer function and the sum of the inner product of the converted second phase-contrast image value and a second transfer function; (d) adding the square sum of the first transfer function and the second transfer function to a noise suppression function; (e) dividing the sum of step (c) by the sum of step (d); and (f) Perform inverse Fourier transform on the result of step (e) to obtain the phase corresponding to each detected position. 10 . The method of claim 9 , wherein the noise suppression function further comprises a high frequency suppression function and a low frequency suppression function. 11 .