阅读说明：本技术 一种基于偏振光栅的点衍射数字全息显微装置及方法 (Point diffraction digital holographic microscopic device and method based on polarization grating ) 是由 郜鹏 温凯 郑娟娟 马英 刘立新 王宇 张美玲 于 2020-04-29 设计创作，主要内容包括：本发明公开了一种基于偏振光栅的点衍射数字全息显微装置及方法,包括激光器、光强控制单元、显微物镜、第一薄透镜、反射镜、光栅、第二薄透镜、针孔滤波器、偏振片和CMOS相机。本发明还公开了一种基于偏振光栅的点衍射数字全息显微方法。该点衍射数字全息显微装置和方法具有稳定性高、可实时振幅/相位成像等优点,可以被广泛应用于生物医学成像、工业检测等多个领域。(The invention discloses a point diffraction digital holographic microscopic device and a method based on a polarization grating. The invention also discloses a point diffraction digital holographic microscopy method based on the polarization grating. The point diffraction digital holographic microscopy device and the method have the advantages of high stability, real-time amplitude/phase imaging and the like, and can be widely applied to multiple fields of biomedical imaging, industrial detection and the like.)

1. A point diffraction digital holographic microscopic device based on a polarization grating is characterized in that: the device comprises a laser (1), a light intensity control unit (2), a sample to be detected (3), a microscope objective (4), a first thin lens (5), a reflector (6), a grating (7), a second thin lens (8), a first pinhole filter (9), a polaroid (10) and a CMOS (complementary metal oxide semiconductor) camera (11), wherein the sample to be detected (3) is placed on a front focal plane of a telescope system consisting of the microscope objective (4) and the first thin lens (5), and an amplified real image appears on a rear focal plane of the system; the grating (7) is placed on the image surface of the sample and is imaged on the CMOS camera (11) by the second thin lens (8), and the grating (7) and the CMOS camera light-sensitive surface satisfy the imaging relation that:here, f 'is the focal length of the second thin lens (8), l is the distance from the grating (7) to the second thin lens (8), and l' is the distance from the CMOS receiving surface to the second thin lens (8); the first pinhole filter (9) is arranged at the back focal plane of the second thin lens (8) and performs pinhole filtering on the-1 st order diffracted light to form a light beam which does not contain object light information any more and is used as reference light.

2. The polarization grating-based point diffraction digital holographic microscopy apparatus of claim 1, wherein: the laser emitted by the laser (1) is linearly polarized light, or the polarization degree is 5: 1, elliptical polarized light.

3. The polarization grating-based point diffraction digital holographic microscopy apparatus of claim 1, wherein: the light intensity control unit (2) consists of a continuously adjustable attenuator and an 1/4 wave plate; when the polarization direction of the polarizer (10) is perpendicular to the polarization direction of the laser (1), the relative light intensities of the object light and the reference light are adjusted by rotating 1/4 wave plates.

4. The polarization grating-based point diffraction digital holographic microscopy apparatus of claim 1, wherein: the grating (7) is a polarization diffraction grating, incident light is diffracted to +/-1 order, the diffraction of the polarization diffraction grating has polarization characteristics, the +/-1 order light intensity has opposite variation trend along with the polarization variation of the incident light, and the period of the grating (7) is less than 2M; where M, the microscope objective (4) and the first thin lens (5) constitute the magnification and spatial resolution of the telescope system, respectively.

5. The polarization grating-based point diffraction digital holographic microscopy apparatus of claim 1, wherein: the first pinhole filter (9) consists of a big hole and a small hole and respectively filters the frequency spectrum of the plus or minus 1-order diffraction light; the big hole does not change the frequency spectrum of the +1 st order diffraction light, and the small hole performs low-pass filtering on the-1 st order diffraction light to form a light beam which does not contain object light information any more and is used as reference light.

6. The polarization grating-based point diffraction digital holographic microscopy apparatus of claim 1, wherein: the polarizing plate (10) makes the +1 order diffracted light and the-1 order diffracted light have the same polarization direction so that the object light and the reference light can interfere with each other.

7. The polarization grating-based point diffraction digital holographic microscopy apparatus of claim 1, wherein: the CMOS camera (11) is used for receiving an interference pattern generated by interference of +1 diffraction light and-1 st order diffraction light; the pixel size of the camera should be smaller than 1/3 where the object light and the reference light form a fringe period.

8. A point diffraction digital holographic microscopy method based on a polarization grating is characterized in that: the method comprises the following steps:

step 1, turning on a laser (1) and a CMOS camera (11), and adjusting a continuous attenuator in a light intensity control unit (2) to enable the light intensity in the CMOS camera (11) to reach a state close to saturation;

step 2, placing a sample (3) to be detected at the front focal plane position of the objective lens (4), and enabling a clear sample image to appear on the CMOS camera (11) by adjusting the axial position of the sample;

step 3, finely adjusting the transverse position of the small hole in the first pinhole filter (9) to ensure that the light intensity of the reference light in the field of view of the camera (11) is uniform; adjusting an 1/4 wave plate in the light intensity control unit (2) to enable the fringe contrast of the holographic pattern in the CMOS camera (11) to reach a maximum value, wherein the light intensities of the object light and the reference light are nearly the same;

step 4, acquiring the holographic pattern of the sample by utilizing a CMOS camera (11)

Step 5, using a formulaAnd reconstructing the recorded off-axis digital hologram by using the method to recover the amplitude and phase distribution information of the sample (3) to be detected.

Technical Field

The invention belongs to the technical field of microscopic imaging, and particularly relates to a point diffraction digital holographic microscopic device and method based on a polarization grating.

Background

Optical microscopy has become an important means for people to observe the micro world since the advent of optical microscopy in the 17 th century. However, the conventional optical microscopy can only obtain the intensity information of the observed sample, and cannot be used for a transparent sample. In order to overcome the defect of low imaging contrast of the traditional optical microscope, the fluorescence microscopy technology utilizes chemical dye or fluorescent protein to specifically mark cells, and can realize high-contrast and selective imaging of specific structures or functional groups of the cells. Therefore, it is increasingly desired to invent an imaging technique that allows cells to be observed in a natural state.

Phase imaging is produced as a unmarked microscopic technology, which utilizes the phase, another important characteristic of light waves except amplitude, not only can observe a transparent sample with high contrast, but also can quantitatively obtain information such as three-dimensional appearance, internal structure, refractive index distribution and the like of an object. However, unlike conventional intensity imaging, the phase distribution is invisible to the naked eye and must be obtained by special imaging means. The Shack-Hartman wavefront sensor can reconstruct the wavefront distribution before passing through the micro-lens array by measuring the offset of each sub-focus after passing through the micro-lens array with distorted wavefront. However, this method is limited by the physical size of the microlenses, and its spatial resolution is low, and thus it is rarely applied to the field of microscopy.

In 1942, zernike proposed that a ring-shaped light source was used to illuminate the sample, and a ring-shaped phase plate was used to delay the phase of the zero-frequency component of the object wave, so as to convert the phase information of the sample into intensity information, and implement phase contrast microscopy. Subsequently, Normarski proposes a differential interference phase contrast microscopy technique, which divides the object light into two parallel portions, which are displaced in a certain direction by a certain distance so as to interfere. The interference pattern reflects the derivative of the measured phase in the shear direction. Both of these microscopic techniques improve the image contrast of the transparent sample under the microscope. However, in the two techniques, there is a non-linear correspondence between the intensity of the image and the phase of the sample, and the true phase distribution of the sample cannot be obtained from the phase contrast image.

Single beam phase microscopy, by imposing certain constraints, converts the phase distribution of a sample into intensity modulation during diffraction. The original phase information can be recovered from the diffraction pattern by a certain algorithm. For example, a phase measurement technique based on a Transmission of Intensity Equation (TIE) can quantitatively acquire phase information of a measured optical wave without iterative operation by recording three Intensity images having different defocus amounts near a focal plane. The method has the advantages of non-interference, simple structure, suitability for partial coherent illumination, no need of phase unwrapping and the like. The spatial domain stacked imaging (psychographic) technology can recover the phase information of a sample from a diffraction image by moving a focused light beam to illuminate different parts of the sample and recording the corresponding diffraction image by using an iterative algorithm. Fourier stacked Microscopy (FPM) technology combines phase retrieval with synthetic aperture by recording low resolution images at different angles and using iterative algorithms to obtain large field of view and high resolution amplitude and phase images. This method does not require a precise mechanical scanning device, but still requires a large number of intensity images to be taken and a complicated calculation process to reproduce the phase, which limits the development of FPM in high-speed and real-time imaging. In general, the accuracy of single beam phase imaging based on optical diffraction constraints is much lower than the phase accuracy of optical interference.

The digital holographic microscopy combines optical interference and optical microscopy, and recovers the amplitude and phase information of the sample to be detected by utilizing the interference between object light and reference light. The technology has high phase measurement precision, and can reach lambda/100 for example. However, this method requires a light source with good coherence to satisfy the coherence condition between the object light and the reference light, and thus brings speckle noise to reduce the imaging quality. In addition, the method needs to introduce an additional reference light path, so that the light path is complex, and the experiment is sensitive to the vibration of the environment. Therefore, digital holographic microscopy imposes particularly stringent requirements on the experimental environment. Popescu provides an off-axis point diffraction phase microscopic method of a physical parameter common path, and realizes real-time quantitative measurement of biological cells. The method divides object light into two parts by a diffraction grating, wherein one part is filtered by a pinhole on a frequency spectrum surface to become reference light, and the other part is still used as the object light; the amplitude and phase information of the measured sample can be reproduced by using the interference pattern between the object light and the reference light. In this optical path, the object light and the reference light experience exactly the same optical path, thus overcoming the effect of environmental disturbances on the measurement results. In 2010, a coaxial point diffraction phase microscopy method based on double gratings is provided, wherein light splitting is realized through a first grating, and phase shifting can be realized by moving a second grating in the vector direction of the gratings. Compared with the traditional off-axis point diffraction phase microscopic method, the method adopts a coaxial light path, and improves the spatial resolution of phase imaging. However, it should be noted that: the existing point diffraction phase microscopy technology forms reference light by filtering object light waves, so that the light intensity of the reference light is related to a detected sample, and the interference patterns of all samples cannot be guaranteed to have high fringe contrast.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a point diffraction digital holographic microscopic device and a method based on a polarization grating, which can be used for measuring the three-dimensional appearance or refractive index distribution of a tiny object. The light path has the advantages of good vibration resistance, capability of carrying out real-time amplitude/phase quantitative microscopic imaging, simple structure and the like.

The technical scheme is as follows:

a point diffraction digital holographic microscopic device based on a polarization grating comprises a laser 1, a light intensity control unit 2, a sample 3 to be measured, a microscope objective 4, a first thin lens 5, a reflector 6, a grating 7, a second thin lens 8, a first pinhole filter 9, a polaroid 10 and a CMOS camera 11, wherein the sample 3 to be measured is placed on a front focal plane of a telescope system consisting of the microscope objective 4 and the first thin lens 5, and an amplified real image appears on a rear focal plane of the system. The grating 7 is placed on the image plane of the sample and imaged by the lens 8 to the CMOS camera11, respectively. At this time, the grating 7 and the photosensitive surface of the CMOS camera satisfy the imaging relationship:here, f 'is the focal length of the second thin lens 8, l is the distance from the grating 7 to the second thin lens 8, and l' is the distance from the CMOS receiving surface to the second thin lens 8. The first pinhole filter 9 is placed at the back focal plane of the second thin lens 8, and performs pinhole filtering on the-1 st order diffracted light to form a light beam no longer containing object light information as reference light.

Further, the laser emitted by the laser 1 is linearly polarized light, or the polarization degree is 5: 1, elliptical polarized light.

Further, the light intensity control unit 2 is composed of a continuously adjustable attenuator and an 1/4 wave plate. When the polarization direction of the polarizing plate 10 is perpendicular to the polarization direction of the laser 1, the relative intensities of the object light and the reference light can be adjusted by rotating 1/4 the wave plate.

Furthermore, the grating 7 is a polarization diffraction grating, which diffracts the incident light to + -1 st order (the light intensity of other diffraction orders is small and can be ignored), and the polarization states of the + -1 st order diffracted light are different (for example, + -1 st order is respectively left-handed circularly polarized light and right-handed circularly polarized light), and the light intensities of the two show opposite variation trends (one is increased, the other is decreased, and vice versa) along with the polarization change of the incident light. In order to ensure that the high frequency components of the object light wave are not lost by the diffracted light, the period of the grating 7 should be less than 2M. Here M, the fiber objective 4 and the first thin lens 5, respectively, constitute the magnification and spatial resolution of the telescope system.

Further, the first pinhole filter 9 is composed of a "large hole" and a "small hole" and filters the spectrum of the ± 1 st order diffracted light, respectively. Wherein the 'big hole' does not change the frequency spectrum of the +1 st order diffracted light (used as object light), and the 'small hole' performs low-pass filtering on the-1 st order diffracted light (only retaining zero-frequency components) to form a light beam no longer containing object light information as reference light.

Further, the polarizing plate 10 makes the +1 diffracted light (object light) and the-1 st order diffracted light (reference light) have the same polarization direction, so that the object light and the reference light can interfere.

Further, the CMOS camera 11 is configured to receive an interference pattern generated by interference of +1 diffracted light (object light) and-1 st diffracted light (reference light). The pixel size of the camera should be smaller than 1/3 where the object light and the reference light form a fringe period.

The invention discloses a point diffraction digital holographic microscopy method based on a polarization grating, which comprises the following steps:

step 1, turning on the laser 1 and the CMOS camera 11, and adjusting a continuous attenuator in the light intensity control unit 2 to enable the light intensity in the CMOS camera 11 to reach a state close to saturation.

And 2, placing the sample 3 to be detected at the front focal plane position of the microscope objective 4, and axially adjusting the axial position of the sample to enable the CMOS camera 11 to display a clear image of the sample.

And 3, finely adjusting the transverse position of the small hole in the first pinhole filter 9 to ensure that the light intensity of the reference light is uniform. The 1/4 wave plate in the light intensity control unit 2 is adjusted so that the fringe contrast of the hologram pattern in the CMOS camera 11 reaches a maximum value when the light intensities of the object light and the reference light are nearly the same.

Step 4, acquiring the holographic pattern of the sample by using the CMOS camera 11

Step 5, using a formulaAnd reconstructing the recorded off-axis digital hologram by using the method to recover the amplitude and phase distribution information of the sample 3 to be detected.

The invention has the beneficial effects that:

the traditional optical microscopy can only acquire the intensity information of an observed sample, and cannot be used for a transparent sample. Digital Holographic Microscopy (DHM), a label-free Microscopy technique, can provide high-contrast phase imaging of live, clear cells in nature. The invention provides a point diffraction digital holographic microscopy method based on a polarization grating. The method not only has the advantage of high measurement precision of the traditional digital holographic microscopic phase, but also has the following advantages: firstly, an optical structure of a parametric common path is adopted, so that the influence of environmental vibration on phase imaging is overcome (the stability is high); secondly, the structure of the object-parameter common path enables the method to adopt a low-coherence light source to inhibit coherent noise; finally, the method utilizes the polarization characteristic of the polarization diffraction grating to adjust the image contrast by rotating a polaroid or a quarter-wave plate, and overcomes the defect of low contrast of the traditional point diffraction phase micro-fringes. In conclusion, the point diffraction digital holographic microscopy device and the point diffraction digital holographic microscopy method have the advantages of high stability, real-time amplitude/phase imaging and the like, and can be widely applied to multiple fields of biomedical imaging, industrial detection and the like.

Drawings

FIG. 1 is a digital holographic microscopic optical path diagram of an objective-parameter common path;

figure 2 is a digital holographic microscopy optical path diagram of an objective path,

FIG. 3 is a diffraction beam splitting of incident light by a polarization grating;

FIG. 4 is a low pass filtering of the-1 diffraction order;

fig. 5 shows the influence of the measured sample on the intensity of the reference light. Wherein, before placing the sample, the light intensity distribution of the object light (O, graph A) and the reference light (R, graph B), and after placing the sample (lily mature anther), the light intensity distribution of the object light (O, graph C) and the reference light (R, graph D);

FIG. 6 shows the adjustment effect of the rotating 1/4 wave plate on the intensity of the object light and the reference light. (a) The average light intensity change curve of object light and reference light in the process of rotating 1/4 wave plates; (b) when the main axis direction and the horizontal direction of the 1/4 wave plate are respectively 0 degrees, 30 degrees, 60 degrees and 90 degrees, the intensity distribution of the object light, the reference light and the interference pattern is obtained;

FIG. 7 imaging results of Lily anthers; (a) a recorded off-axis digital hologram; (b) IR_DSpectral distribution and window function ofSelected regions (shown in dashed lines); (c) and (d) the reproduced intensity and phase distribution;

FIG. 8 imaging results of breast cancer cells of mice; (a) a reproduced amplitude image; (b) reproduced phase image

Figure 9 stability testing of the device itself. (a) Phase distribution (rad) of the device itself without sample placed; (b) the phase of a point in the field of view (shown in black boxes) is plotted against time. (a) The scale in (1): 10 mu m; phase jump profile pitch: 1 radian.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Referring to fig. 1, a polarization grating-based point diffraction digital holographic microscopy apparatus includes the following devices:

description of the devices used:

1. the laser 1 has a wavelength in the visible light range, and is required to have a suitable coherence length because of stable output laser power.

2. The light intensity control unit 2 is composed of a continuously adjustable attenuator and an 1/4 wave plate.

3. The first thin lens 5 and the second thin lens 8 are required to be achromatic double cemented lenses.

4. A first pinhole filter 9 for limiting the illumination divergence angle of the illumination light.

5. And the microscope objective 4 is used for expanding the beam of the sample and realizing microscopic imaging.

6. The grating 7 is a polarization diffraction grating, and diffracts the incident light to ± 1 st order, and the ± 1 st order diffracted lights are left circularly polarized light and right circularly polarized light, respectively. The grating has high diffraction efficiency at the order of +/-1.

7. A second pinhole filter 13. In order to facilitate installation and fixation, a small hole and a large hole can be machined in one metal sheet. The aperture, i.e., pinhole filter, should have a suitable diameter (e.g., 25 μm) for low pass filtering the-1 st order diffracted light, and should ensure that the intensity of the filtered beam is approximately uniform across the CCD field of view. For this purpose reference may be made to: the diameter D is less than or equal to 1.22 lambda f/D. Where λ is the wavelength of the illumination light, f is the focal length of the second thin lens 8, and D is the diameter of the light beam before the second thin lens 8. The large holes are used to pass all the +1 order diffracted light spectrum.

8. The polarizing plate 10 causes the +1 diffracted light (object light) and the-1 st diffracted light (reference light) to have the same polarization direction and to interfere with each other.

CMOS camera 11, normal black and white CMOS, with appropriate grey scale, pixel size and number of pixels.

The optical path proposed by the invention is shown in fig. 1. The laser light emitted from the laser 1 is used as illumination light after passing through a continuously adjustable attenuator and an 1/4 wave plate. The sample 3 to be measured is placed in the front focal plane of the telescopic system consisting of the microscope objective 4 and the first thin lens 5, so that an enlarged real image will appear in the back focal plane of the system. The polarization grating 7 is placed on the image surface, and divides the object light into two identical light waves which propagate along the +/-1 diffraction order direction through diffraction. As shown in fig. 3, the ± 1 diffracted lights are left-circularly polarized light and right-circularly polarized light, respectively, and the intensity of the ± 1 diffracted light thereof is related to the polarization direction of the incident light. The grating 7 and the real image of the sample are imaged by the second lens 8 onto the CMOS camera 11 face. As shown in fig. 4, the spectra of two object lights propagating along the ± 1 diffraction directions appear on the back focal plane of the second lens 8. The +1 st order diffracted light passes through the large hole of the first pinhole filter 9, and the spectrum is not affected and used as object light. The-1 st order diffracted light is filtered by the pinholes in the first pinhole filter 9 to become spherical light (no longer carrying object light information) which is used as reference light. After passing through the polarizer 10, the object reference light has the same polarization direction and interferes. The CMOS camera 11 is placed on an image plane where the grating is imaged by the second lens 8 to receive the interference pattern of the object light and the reference light. The relative intensities of the object light and the reference light can be adjusted by rotating the quarter-wave plate. In summary, the following steps: since the object light and the reference light are subjected to identical optical elements, the device has less influence on the vibration of the environment.

The original optical path is in a linear trend, and the structure is simple but the occupied space is large. In order to reduce the size of the device, a mirror 6 may be added to the original optical path to fold the optical path, as shown in fig. 1. The folded optical path also has the advantage of an inverted microscope, such as facilitating the observation of adherent cells in a culture dish from below. In addition, when the light beam emitted by the laser is small, a beam expanding and collimating system can be added to expand the light beam. As shown in fig. 2, the beam expanding system is composed of a thin first lens 12 and a second lens 14, and a second pinhole filter 13 is disposed on the back focal plane of the first lens 12 to perform low-pass filtering on the light beam for improving the quality of the illumination light beam. Compared with the physical parameter common path digital holographic microscopic light path diagram in the figure 1, a beam expanding system is additionally added. The beam expanding system comprises a first lens 12, a second pinhole filter 13 and a second lens 14. The second pinhole filter 13 here is to limit the illumination divergence angle of the illumination light to further improve the beam quality.

The principle of the invention is as follows:

the light wave emitted by the laser 1 is linearly polarized along the horizontal direction and is denoted asWhen the incident light passes through the quarter-wave plate (2) with the main axis direction forming an angle theta with the horizontal direction, the polarization state can be represented as follows:

after passing through the sample, the illumination light forms object light waves which are imaged by a microscopic amplification system (4-5) onto a polarization grating 7. The polarization grating divides the object light into a plurality of parts through diffraction, and the object light is respectively transmitted along the directions of different orders of diffraction light. Wherein, the light intensity ratio of + -1 order diffraction light is more than 40%, which are respectively used as object light and reference light (formed by performing spectrum filtering on-1 diffraction light). The diffraction orders other than the ± 1 st order diffracted light are all blocked at the focal plane of the second thin lens 8. In addition, the polarization grating has excellent polarization diffraction characteristics: the + -1 st order diffracted light has different polarization states, and the relative intensity of the two is related to the polarization state of the incident light. We theoretically analyze the polarization diffraction characteristics by using the jones matrix, and the complex transmittance of the polarization grating can be expressed as:

here, τ_oAnd τ_eRespectively, the transmittances of o light and e light, Δ n_oAnd Δ n_eRespectively representing the refractive indexes of the grating for o light and e light; d (x, y) represents the grating thickness distribution;representing the spatial phase modulation function of the polarization grating for o and e, respectively. Where k is₀＝2π/λ,Ψ_e(x, y) represents the direction of the principal axis of anisotropy as a function of space. We can get the followingRespective polarization components ofExpansion into a Fourier series:where i and j take values of 1 and 2, l is an integer representing the diffraction order, K2 pi/Λ is the grating vector, Λ is the grating period, then the transfer matrix for the light wave propagating along the +1 order of the grating can be expressed as:

the transfer matrix propagating along the grating-1 order can be expressed as:

when an incident beam a passes through the sample (forming object light O (x, y)) and is diffracted by this grating, ± 1 st order diffracted light can be expressed as:

here the constant term is ignoredA_OShowing the complex amplitude distribution of the illumination light a after passing through the sample. After diffraction of grating, along +1Order-wise propagation (used as object light), 1 st order diffracted light D_-1After being filtered by the pinhole, the complex amplitude is changed into A_RThe polarization direction of the reference light is unchanged. When the object light and the reference light pass through the polarizing plate 10 and become linearly polarized light with the same polarization direction, the object light and the reference light interfere with each other on the CCD plane. Specifically, when the polarization direction of the polarizer (10) is at 45 ° to the x-axis, its transmittance function can be written as:

the complex amplitudes of the object light and the reference light after passing through the polarizing plate (10) can be expressed as:

for the polarization grating we have chosen to be,in this case, formula (7) can be written as:

as can be seen from the formula (8),andis related to the polarization state of the incident light (determined by the orientation of the major axis of the 1/4 wave plate). As can be seen from the view in figure 6,andthe relative light intensity of (1) shows an opposite trend along with the change rule of theta (b) ((b))When the size of the pipe is increased, the pipe is enlarged,reduced), cos predicted in accordance with formula (8)²Theta and sin²The intensity relationship of theta.

On the surface of the CCD, the light source is arranged,andinterference occurs, and its intensity distribution can be expressed as:

k represents the carrier frequency content of the interference fringes on the CCD plane,representing the phase difference between the object light and the reference light.

By adopting the traditional off-axis digital holographic reconstruction method, the complex amplitude of the object light can be reconstructed

Here, d₀The defocusing distance is shown, namely the distance from the CCD to the image surface of the sample; r_DExp (-iKx) is a digital reference light, which can be determined by measuring the carrier frequency quantity K' of the fringes. I R_DFT {. and IFT {. can be used to compensate for spectral shifts caused by the angle between the object and reference light, respectively, Fourier transform and inverse Fourier transform (ξ) represent coordinates in the frequency domain.For a window function, in thatThe value of the region (1) is 1, and the values of other regions are 0, so that the spectral distribution of the object light real image is selected. Using the reproduced complex amplitude O_r(x,y,d₀) And relationThe amplitude image | O of the sample can be obtained_r(x, y) | and phase imageFinally, byThe three-dimensional shape d (x, y) and the refractive index distribution of the tested sample can be calculated

As described above, the conventional point diffraction phase microscopy forms the reference light by filtering the object light wave, and thus the intensity of the reference light is related to the specific sample to be measured. To verify this, in fig. 5, we measured the intensity of the object and reference light propagating along the ± 1 diffraction directions before and after placing the sample, respectively. The first row in fig. 5 shows the intensity distribution of the object light (O, left) and the reference light (R, right) before placing the sample. The second row in fig. 5 shows the intensity distribution of the object light (O, left) and the reference light (R, right) after placing the sample (lily mature anther). Comparing the upper and lower rows of images to know that: when the spatial structure of the sample is relatively complex, the intensity of the reference light (generated by pinhole filtering) is relatively small. This is because: when the sample structure is complicated, the energy of the object light is mostly distributed in high-frequency components, and does not contribute to the light intensity of the reference light.

Meanwhile, as can be seen from the formula (8): by rotating the direction of the principal axis of the quarter-wave plate, the relative intensity between the object light and the reference light can be adjusted to optimize the contrast of the interference pattern. To verify this, we rotated the principal axis direction of the 1/4 wave plate to different angles (relative to the horizontal direction) and rotated the polarizer 10 to the vertical direction in experiments, and measured the intensity distributions of the object light, the reference light, and the interference pattern in different cases, as shown in fig. 6, thereby obtaining the effect of optimizing the fringe contrast of the interference pattern. When the incident light direction is the horizontal direction (and the polarization direction of the polarizing plate 10 is in the horizontal direction), by rotating the principal axis direction of the 1/4 wave plate, the ratio of the light intensities of O and R can be changed from 1: 6, adjusting to 6: 1. therefore, by adjusting 1/4 the wave plate to make the intensity of the object light and the reference light the same (e.g., 1/4 the wave plate is located at 30 °), the contrast of the interference pattern reaches the maximum.