A fast quantitative differential interference microscopy system and method based on holographic recording

Abstract

This invention discloses a rapid quantitative differential interferometry microscopy system and method based on holographic recording. The system includes a partially coherent illumination module and, sequentially arranged along the optical axis of the partially coherent illumination module, a microscope objective, a microscope tube lens, a transmission grating, a first linear polarizer, a first thin lens, a half-wave plate, a light-shielding plate, a phase-type spatial light modulator, a second thin lens, a second linear polarizer, and an image acquisition module. This invention utilizes a phase-type spatial light modulator to achieve shear shifts in any direction and to any degree, making it suitable for various applications. The system uses the grating carrier frequency to achieve holographic recording of differential interferometry, enabling quantitative differential interferometry detection of the sample using only two acquired images. It exhibits very high temporal resolution, spatial resolution, and imaging accuracy, showing broad application prospects in biological imaging and industrial detection.

Description

Technical Field

[0001] This invention belongs to the field of optical microscopy imaging technology, specifically relating to a rapid quantitative differential interferometry microscopy system and method based on holographic recording. Background Technology

[0002] In today's era of continuous advancements in science and technology, quantitative phase microscopy (QPM), an emerging technology that enables high-resolution and high-contrast detection without requiring sample pretreatment, is making remarkable progress. QPM achieves high-contrast imaging of transparent samples by recovering the phase change caused by illumination light passing through the sample. It is a label-free and non-invasive imaging method, and the detection process does not damage or affect the sample. QPM plays a crucial role in biomedicine, microbiology, materials science, drug development, and other fields, bringing significant assistance and breakthroughs to scientific research and practical applications.

[0003] Quantitative differential interferometry (QDIS) microscopy is an advanced quantitative phase microscopy technique characterized by high stability, high imaging quality, and high axial resolution, making it widely used in biological imaging and industrial inspection. QDIS imaging can be achieved through various methods. Gradient light QDIS microscopy, which couples a phase-type spatial light modulator to a traditional differential interferometric phase-contrast microscope, can provide high-quality quantitative phase imaging of thick tissue samples. However, it is difficult to accurately determine the shear shift during implementation, significantly impacting imaging accuracy. Laser structured light-based QDIS microscopy utilizes a digital grating for structured illumination of the sample, followed by imaging at a specific defocused surface to achieve differential interferometric detection. To achieve quantitative phase imaging, this method combines a four-step phase-shift operation and angular spectral transfer technology. However, the defocusing process makes it difficult to accurately determine the shear shift and sample defocusing amount, thus compromising imaging accuracy.

[0004] Quantitative differential interferometry (QDIS) based on pupil-plane amplitude gratings (amplitude-type spatial light modulators) falls into two categories. The first method utilizes the 0th and ±1st order diffraction terms of the amplitude grating for differential interferometry detection, followed by a four-step phase-shifting operation to obtain the sample's phase distribution. This technique requires the amplitude gradient of the sample to be zero everywhere, thus significantly limiting its application. The second method uses the +1st order diffraction term of a dual-frequency amplitude grating for differential interferometry detection, followed by a three-step phase-shifting operation to obtain the sample's phase distribution. In this technique, the spatial light modulator needs to simultaneously load two grating patterns with very small differences in their periods. Therefore, this technique requires complex random coding techniques to achieve differential interferometry detection. Furthermore, this technique requires a laser with a very narrow linewidth as the light source, and image quality is negatively affected by laser speckle. The latest QDIS technique utilizes the polarization modulation characteristics of a phase-type spatial light modulator for differential interferometry detection, significantly improving the performance of QDIS. However, this technique requires four phase-shifting operations (8 original images) to achieve quantitative phase imaging of the sample under test, which greatly limits the imaging speed.

[0005] The aforementioned quantitative differential interferometry (QDIS) microscopy techniques all require phase-shifting operations to achieve quantitative phase imaging, necessitating the acquisition of multiple phase-shift intensity maps. This significantly limits imaging speed, making it difficult to capture rapid dynamic changes. To improve the imaging speed of QDIS, researchers have proposed several methods. First, in QDIS based on pupil-plane amplitude grating modulation, researchers achieved single-exposure collection of multiple differential interferometric phase-shift maps by limiting the imaging field of view and adding complex multi-frequency amplitude gratings. This makes the imaging speed of QDIS limited only by the camera's exposure time. However, this method requires a laser with a very narrow linewidth as the illumination source, and image quality is adversely affected by laser speckle. Furthermore, the alignment between multiple differential interferometric phase-shift maps directly affects the imaging accuracy of this technique. Second, QDIS based on four-wave shearing achieves rapid single-exposure quantitative phase imaging by placing a two-dimensional amplitude grating at a certain position in front of the imaging plane. However, this technique requires mechanically adjusting the axial distance of the two-dimensional amplitude grating relative to the imaging plane to achieve differential interferometry detection. The shear offset is difficult to determine precisely, thus hindering the accuracy of the imaging. Recently, researchers achieved rapid quantitative phase imaging based on differential interferometry in a single exposure by placing a Wollaston prism on the imaging plane of a conventional differential interferometry phase-contrast microscope. This method is simple in structure, highly stable, and produces high-quality images, with the imaging speed only affected by the camera's exposure time. However, this method requires the shear plane of the Wollaston prism to be strictly coincident with the imaging plane of the conventional differential interferometry phase-contrast microscope, and the shear offset direction caused by the Wollaston prism to be strictly consistent with the shear offset direction caused by the conventional differential interferometry phase-contrast microscope. Therefore, the practical implementation of this technique faces significant challenges. Furthermore, the shear offset induced by this technique is difficult to determine precisely, making its widespread application in practice difficult. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention provides a rapid quantitative differential interferometry microscopy system and method based on holographic recording. The technical problem to be solved by this invention is achieved through the following technical solution:

[0007] One aspect of the present invention provides a rapid quantitative differential interferometry microscopy system based on holographic recording, comprising a partially coherent illumination module and, sequentially arranged along the optical axis of the partially coherent illumination module, a microscope objective, a tube lens, a transmission grating, a first linear polarizer, a first thin lens, a half-wave plate, a phase-type spatial light modulator, a second thin lens, a second linear polarizer, and an image acquisition module, wherein...

[0008] The partially coherent illumination module is used to generate partially coherent illumination light; the sample is placed at the front focal plane of the microscope objective, and the transmission grating is placed at the confocal plane of the tube lens and the first thin lens; the first linear polarizer is placed between the transmission grating and the first thin lens; the polarization direction of the first linear polarizer is along the Y-axis, and the first thin lens only collects the 0th and ±1st order diffracted light carrying object light wave information and polarized along the Y-axis;

[0009] The phase-type spatial light modulator is disposed at the confocal surface of the first thin lens and the second thin lens, and the working surface of the phase-type spatial light modulator only covers the spectral distribution of the 0th and -1st order diffracted light. The half-wave plate is disposed between the first thin lens and the phase-type spatial light modulator and is adjacent to the working surface of the phase-type spatial light modulator, which enables the spectral distribution of the -1st order diffracted light reaching the working surface of the phase-type spatial light modulator to be linearly polarized along the X-axis direction. The X-axis direction and the Y-axis direction are perpendicular to each other and both are perpendicular to the optical axis direction of the partially coherent illumination module.

[0010] The phase-type spatial light modulator can perform phase modulation of the light field with polarization direction selectivity; the second linear polarizer is disposed between the second thin lens and the image acquisition module, and is used to adjust the polarization of the 0th order diffracted light and the -1st order diffracted light with the same polarization direction; the image acquisition module is used to acquire and record images.

[0011] Another aspect of the present invention provides a rapid quantitative differential interferometry microscopy method based on holographic recording, comprising:

[0012] S1: Obtain the light intensity distribution of the sample using the rapid quantitative differential interferometry microscopy system described in any of the above embodiments;

[0013] S2: Perform a spatial Fourier transform on the light intensity distribution to obtain the spectral distribution of the light intensity;

[0014] S3: Based on the spectral distribution of the light intensity, the phase distribution of the sample is obtained using the holographic differential reconstruction method.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention proposes a rapid quantitative differential interferometry microscopy system and method based on holographic recording, which has the following advantages: First, the optical structure of partially coherent illumination and coaxial interference enables the device to simultaneously possess high spatial phase sensitivity, high temporal phase sensitivity, high imaging stability, and high imaging quality. Second, the microscopy system utilizes a phase-type spatial light modulator to precisely achieve shear shifts in any direction and to any degree, resulting in very high imaging accuracy, suitable for various application scenarios. Furthermore, the microscopy system utilizes a grating carrier frequency to achieve holographic recording of differential interference, enabling high-precision quantitative differential interferometry imaging of the sample under test using only two images, exhibiting very high temporal and spatial resolution, as well as very high imaging accuracy. Finally, the microscopy system has a simple structure, is easy to assemble, and inherits the high axial resolution characteristic of differential interferometry phase-contrast microscopy, enabling three-dimensional quantitative phase imaging of thick tissue samples. Therefore, the proposed rapid quantitative differential interferometry microscopy system based on holographic recording can perform label-free, highly stable, highly sensitive and high-resolution in-situ quantitative phase imaging of the sample to be tested, and has very good scalability in terms of structure and function, and has great application value in the fields of biomedicine and industrial detection.

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the structure of a rapid quantitative differential interferometry microscopy system based on holographic recording provided in an embodiment of the present invention;

[0019] Figure 2 This is a schematic diagram of the layout of a half-wave plate on the pupil surface, a phase-type spatial light modulator, and a light-shielding plate provided in an embodiment of the present invention;

[0020] Figure 3 This is a simulation experiment of the true phase distribution of a sample to be tested, provided by an embodiment of the present invention.

[0021] Figure 4 It is along Figure 3 Phase distribution diagram of the horizontal white dashed lines in the image;

[0022] Figure 5 It is a phase distribution map of a phase-type spatial light modulator that has been sheared and shifted along the X-axis.

[0023] Figure 6 It is a holographic differential interference intensity map that is sheared and offset along the X-axis direction, acquired by the image acquisition module;

[0024] Figure 7 yes Figure 6 The spectral distribution of the holographic differential interference intensity map with shear offset along the X-axis direction;

[0025] Figure 8 Yes Figure 7 The spectrum distribution shown is obtained after shifting and filtering.

[0026] Figure 9 Yes Figure 8 The phase gradient distribution along the X-axis is obtained by performing a two-dimensional inverse Fourier transform on the spectral distribution shown.

[0027] Figure 10 It is a phase distribution map of a phase-type spatial light modulator that has been sheared and shifted along the Y-axis.

[0028] Figure 11 It is a holographic differential interference intensity map that is sheared and offset along the Y-axis direction, acquired by the image acquisition module;

[0029] Figure 12 It is the spectral distribution of the holographic differential interference intensity map that is sheared and offset along the Y-axis;

[0030] Figure 13 Yes Figure 12 The spectrum distribution shown is obtained after shifting and filtering.

[0031] Figure 14 Yes Figure 13 The phase gradient distribution along the Y-axis is obtained by performing a two-dimensional inverse Fourier transform on the spectral distribution shown.

[0032] Figure 15 The phase distribution is recovered using the phase gradient distribution along the X-axis and the phase gradient distribution along the Y-axis.

[0033] Figure 16 It is along Figure 15 Phase distribution of the horizontal white dashed line;

[0034] Figure 17 It is the phase gradient distribution of COS7 cells along the X-axis;

[0035] Figure 18 It is the phase gradient distribution of COS7 cells along the Y-axis;

[0036] Figure 19 The phase distribution of COS7 cells is recovered by using the phase gradient distribution along the X-axis and the phase gradient distribution along the Y-axis of COS7 cells;

[0037] Figure 20 Is with Figure 19 Bright-field images of COS7 cells in the same field of view.

[0038] Explanation of reference numerals in the attached figures:

[0039] 1-Partial coherent illumination module; 2-Sample; 3-Microscope objective; 4-Bullet lens; 5-Transmission grating; 6-First linear polarizer; 7-First thin lens; 8-Half-wave plate; 9-Light shield; 10-Phase spatial light modulator; 11-Second thin lens; 12-Second linear polarizer; 13-Image acquisition module. Detailed Implementation

[0040] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description, in conjunction with the accompanying drawings and specific embodiments, provides a fast quantitative differential interference microscopy system and method based on holographic recording proposed in accordance with the present invention.

[0041] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a more in-depth and concrete understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the technical solutions of the present invention.

[0042] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations are intended to cover non-exclusive inclusion, such that an article or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or apparatus that includes said element.

[0043] Example 1

[0044] Please see Figure 1 , Figure 1This is a schematic diagram of a rapid quantitative differential interferometry microscopy system based on holographic recording, provided in an embodiment of the present invention. The rapid quantitative differential interferometry microscopy system includes a partially coherent illumination module 1 and, sequentially arranged along the optical axis of the partially coherent illumination module 1, a microscope objective 3, a microscope tube lens 4, a transmission grating 5, a first linear polarizer 6, a first thin lens 7, a half-wave plate 8, a phase-type spatial light modulator 10, a second thin lens 11, a second linear polarizer 12, and an image acquisition module 13. The partially coherent illumination module 1 generates partially coherent illumination light; the sample 2 is positioned at the front focal plane of the microscope objective 3; the transmission grating 5 is positioned at the confocal plane of the microscope tube lens 4 and the first thin lens 7; the first linear polarizer 6 is positioned between the transmission grating 5 and the first thin lens 7; the polarization direction of the first linear polarizer 6 is along the Y-axis; the first thin lens 7 collects only the 0th and ±1st order diffracted light carrying object light wave information and polarized along the Y-axis; the phase-type spatial light modulator 10 is positioned along the first thin lens 7. At the confocal surface of lens 7 and the second thin lens 11, and with the working surface of the phase-type spatial light modulator 10 covering only the spectral distribution of the 0th and -1st order diffracted light, a half-wave plate 8 is disposed between the first thin lens 7 and the phase-type spatial light modulator 10 and is adjacent to the working surface of the phase-type spatial light modulator 10. This enables the spectral distribution of the -1st order diffracted light reaching the working surface of the phase-type spatial light modulator 10 to be linearly polarized along the X-axis direction. The X-axis direction and the Y-axis direction are perpendicular to each other and both perpendicular to the optical axis direction of the partially coherent illumination module 1. The phase-type spatial light modulator 10 can perform phase modulation of the light field with polarization direction selectivity. A second linear polarizer 12 is disposed between the second thin lens 11 and the image acquisition module 13 for polarization adjustment of the 0th order diffracted light and the -1st order diffracted light with the same polarization direction. The image acquisition module 13 is used for image acquisition and recording.

[0045] Specifically, in this embodiment, the partially coherent illumination module 1 consists of multiple light-emitting diodes (LEDs) evenly distributed in a ring, each LED having the same spectral bandwidth. The microscope objective 3 and the lens barrel 4 form a confocal system. The lens barrel 4 and the first thin lens 7 also form a confocal system, and a transmission grating 5 is placed at the confocal surface of the lens barrel 4 and the first thin lens 7 to modulate the low-pass filtered object light wave. The quasi-plane wave emitted by each LED of the partially coherent illumination module 1 illuminates the sample 2 located at the front focal plane of the microscope objective 3 at a large angle, thereby generating an object light wave carrying sample information. After magnification and imaging by the confocal system composed of the microscope objective 3 and the lens barrel 4, the object light wave propagates to the confocal surface of the lens barrel 4 and the first thin lens 7. During this process, the object light wave undergoes two spatial Fourier transforms by the microscope objective 3 and the lens barrel 4, and its spectral distribution is limited by the finite aperture of the microscope objective 3. Therefore, the object light wave propagating to the confocal surface of the barrel lens 4 and the first thin lens 7 is actually the result of low-pass filtering. Simultaneously, a transmission grating 5 placed at the confocal surface of the barrel lens 4 and the first thin lens 7 modulates the low-pass filtered object light wave. Under the beam-splitting effect of the transmission grating 5, multiple diffracted beams carrying object light wave information are generated at the front focal surface of the first thin lens 7. Because the first thin lens 7 has a finite aperture, only the 0th and ±1st order diffracted beams are collected by the first thin lens 7.

[0046] Since the light emitted by the partially coherent illumination module 1 is unpolarized, but the subsequent optical path requires polarization modulation of the light field, a first linear polarizer 6 is placed between the transmission grating 5 and the first thin lens 7. The polarization direction of the first linear polarizer 6 is along the Y-axis; therefore, the 0th and ±1st order diffracted light entering the first thin lens 7 are both linearly polarized along the Y-axis. Specifically, after the spatial Fourier transform of the 0th and ±1st order diffracted light carrying object wave information, their spectral information is uniformly distributed at the confocal surface of the first thin lens 7 and the second thin lens 11. It is worth noting that the spectral distribution of the 0th and ±1st order diffracted light at the confocal surface of the first thin lens 7 and the second thin lens 11 presents as three identical circular regions, and they do not overlap with each other. Figure 2 As shown.

[0047] Meanwhile, the phase-type spatial light modulator 10 is placed at the confocal surface of the first thin lens 7 and the second thin lens 11, and its working surface only covers the spectral distribution of the 0th and -1st order diffracted light, such as... Figure 2 As shown. The spectral distribution of the +1st order diffracted light is blocked by the light-shielding plate 9 located at the confocal surface of the first thin lens 7 and the second thin lens 11, preventing it from entering the subsequent optical path. The spectral distribution of the -1st order diffracted light is modulated by the half-wave plate 8 before reaching the phase-type spatial light modulator 10, as shown. Figure 1 and 2As shown. The half-wave plate 8 is placed as close as possible to the working surface of the phase-type spatial light modulator 10, and it only covers the spectral distribution of the -1st order diffracted light. Simultaneously, the fast axis direction of the half-wave plate 8 is aligned with the positive X-axis direction (e.g., ...). Figure 1 (from the paper outwards) and the positive Y-axis direction (e.g.) Figure 1 The light is simultaneously at a 45-degree angle (with the plane of the paper upwards). Therefore, the spectral distribution of the -1st order diffracted light reaching the working surface of the phase-type spatial light modulator 10 is linearly polarized only along the X-axis. Simultaneously, the spectral distribution of the 0th order diffracted light reaching the working surface of the phase-type spatial light modulator 10 is linearly polarized only along the Y-axis. The phase-type spatial light modulator 10 exhibits polarization direction selectivity in its modulation of the light field. In this invention, the phase-type spatial light modulator only affects linearly polarized light along the Y-axis and not linearly polarized light along the X-axis. Therefore, the phase-type spatial light modulator 10 only performs phase modulation on the spectral distribution of the 0th order diffracted light and not on the spectral distribution of the -1st order diffracted light. Finally, the spectral distributions of the modulated 0th order diffracted light and the unmodulated -1st order diffracted light are coherently superimposed on the image acquisition module 13 and recorded by it after undergoing a spatial Fourier transform by the second thin lens 11. It should be noted that the second linear polarizer 12 is placed between the second thin lens 11 and the image acquisition module 13, and its polarization direction is at 45 degrees to both the positive Y-axis and the negative X-axis, so that the 0th order diffracted light and the -1st order diffracted light reaching the image acquisition module 13 have the same polarization direction.

[0048] Preferably, in this embodiment, the wavelength range of the light-emitting diode in the partially coherent illumination module 1 is 488±10 nm, and the power is 60 mW; the microscope objective 3 is an oil-immersion objective with a magnification of 100X and a numerical aperture NA = 1.44 (plan achromatic objective); the focal length of the tube lens 4 is 200 mm; the grating constant of the transmission grating 5 is 8.47 μm; the wavefront distortion of the light wave after passing through the first linear polarizer 6 and the second linear polarizer 12 is less than 1 / 4 of the wavelength; the first thin lens 7 has a focal length of 200 mm. The lens is a cemented doublet achromatic lens with a focal length of 250 mm; the half-wave plate 8 has an achromatic function; the light-shielding plate 9 is made of opaque light-shielding paper; the phase modulation resolution of the phase-type spatial light modulator 10 is 8 bits, its liquid crystal switching time is 2 milliseconds, the pixel range is 1920×1152, and the size of a single pixel is 9.2 μm×9.2 μm; the second thin lens 11 is a cemented doublet achromatic lens with a focal length of 250 mm; the image acquisition module 13 is selected as a CMOS camera, and the size of a single pixel is 2.4 μm×2.4 μm.

[0049] Furthermore, the partially coherent illumination module 1 of this invention is composed of light-emitting diodes with a certain spectral bandwidth, whose center wavelength is denoted as λ. For ease of calculation, let the axis of the rapid quantitative differential interferometry microscopy system of this embodiment be the Z-axis (the horizontal direction to the left in the paper is the positive direction), the intersection of the system axis and the sample surface be the origin O, the direction perpendicular to the paper and outwards is the positive X-axis, and the direction vertically upwards in the paper is the positive Y-axis. Let the focal length and numerical aperture of the microscope objective 3 be f and f, respectively. obj And NA; the focal length of the barrel lens 4 is f. tube The grating constant (period) of the transmission grating 5 is d; the focal length of the first thin lens 7 is f1; the focal length of the second thin lens 11 is f2; the pixel size of the image acquisition module 13 is pixelsize; the width and length of the working surface of the phase-type spatial light modulator 10 are SLM_width and SLM_height, respectively.

[0050] The sample 2, located at the front focal plane of the microscope objective 3, generates an object beam carrying sample information under the illumination of the partially coherent illumination module 1, which is represented as follows: Where (x,y) represents the horizontal spatial coordinates, and a(x,y) represents the amplitude information of the sample. This represents the phase information of the sample. The object light wave undergoes a spatial Fourier transform by the microscope objective 3, filtering by the pupil aperture of the microscope objective 3, and a spatial Fourier transform by the tube lens 4. At the confocal surface of the tube lens 4 and the first thin lens 7, a low-pass filtered object light wave is generated, and its light field distribution is expressed as follows:

[0051]

[0052] in, The convolution operation is represented by E(x,y); E(x,y) represents the ideal light field distribution produced at the rear focal plane of the microscope lens 4 after the object light wave s(x,y) passes through the confocal system composed of microscope objective 3 and microscope lens 4, i.e., the ideal magnified image of s(x,y); h(x,y) is the complex amplitude point spread function of the confocal system composed of microscope objective 3 and microscope lens 4, which represents the light field distribution produced at the rear focal plane of the microscope lens 4 after an infinitesimal point source at the front focal point of microscope objective 3 passes through the confocal system composed of microscope objective 3 and microscope lens 4. The complex amplitude point spread function h(x,y) is also the system coherence transfer function (the pupil function of microscope objective 3). CTF(ξ,η) is the light field distribution produced at the rear focal plane of the microscope lens 4 after the spatial Fourier transform of the microscope lens 4. Here, (ξ,η) represents the spectral coordinates corresponding to the spatial coordinates (x,y). CTF(ξ,η) is only affected by ξ. 2 +η 2 ≤(NA / λ) 2 It is valid within the range of , and ideally, it is all 1 within the valid range.

[0053] Despite s L (x,y) is the convolution between the ideal light field distribution E(x,y) and the complex amplitude point spread function h(x,y). Its spatial resolution is limited by the finite aperture of the microscope objective 3, but it still accurately carries the phase and amplitude information of sample 2. Meanwhile, a two-dimensional transmission grating 5 with a grating constant of d is placed at the confocal surface of the tube lens 4 and the first thin lens 7, with its diffraction direction along the Y-axis. Theoretically, under the beam splitting effect of the transmission grating 5, multiple diffracted light carrying object wave information will be generated at the front focal surface of the first thin lens 7. However, the first thin lens 7 has a finite aperture; in the rapid quantitative differential interference microscopy system of this invention, only the 0th and ±1st order diffracted light can enter the first thin lens 7. Therefore, the modulation function of the light field by the transmission grating 5 can be expressed as g(x,y)=1+cos(2πy / d). Therefore, after the light field distribution shown in formula (1) is modulated by the transmission grating 5, a new light field is generated at the confocal surface of the barrel lens 4 and the first thin lens 7, which is expressed as:

[0054] e g (x,y)=s L (x,y)·g(x,y) (2)

[0055] Since the polarization direction of the first linear polarizer 6 is along the Y-axis, the light field distribution shown in equation (2) can be further expressed using the Jones vector as follows:

[0056]

[0057] Subsequently, after the spatial Fourier transform of the first thin lens 7, the spectral information of the light field distribution shown in formula (3) is distributed at the confocal surface of the first thin lens 7 and the second thin lens 11. As mentioned above, this spectral distribution at the confocal surface of the first thin lens 7 and the second thin lens 11 presents as three identical circular regions, which are uniformly distributed along the Y-axis and do not overlap with each other. Among them, the spectral distribution of the +1st order diffracted light is blocked by the light-shielding plate 9 located at the confocal surface of the first thin lens 7 and the second thin lens 11 and cannot enter the subsequent light path. Therefore, the spectral distribution at the confocal surface of the first thin lens 7 and the second thin lens 11 is expressed as:

[0058]

[0059] Where (ξ,η) represents the spectral coordinates corresponding to the spatial coordinates (x,y), and ~ represents the two-dimensional Fourier transform of the corresponding variable, for example, s L The two-dimensional Fourier transform of (x,y). It should be noted that... in, Let P(ξ,η) represent the two-dimensional Fourier transform of the ideal light field distribution E(x,y), and let P(ξ,η) represent the transform of the light field distribution only in space. A circular function valid within a certain range, whose values ​​are all 1 within the valid range and all 0 outside the valid range, where f obj f represents the focal length of microscope objective 3. tube This indicates the focal length of lens 4 in the telescope.

[0060] Furthermore, in order to modulate the spectral distribution shown in formula (4), the phase-type spatial light modulator 10 is placed at the confocal surface of the first thin lens 7 and the second thin lens 11, and its working surface only covers the spectral distribution of the 0th and -1st order diffracted light, such as... Figure 1 and Figure 2 As shown. Simultaneously, a half-wave plate 8 is placed before the phase-type spatial light modulator 10, and is adjacent to the working surface of the phase-type spatial light modulator 10. The half-wave plate 8 only covers the spectral distribution of the -1st order diffracted light, and is used to modulate the spectral distribution of the -1st order diffracted light. The fast axis direction of the half-wave plate 8 forms a 45-degree angle with both the positive X-axis and the positive Y-axis. Therefore, after modulation by the half-wave plate 8, the spectral distribution of the -1st order diffracted light reaching the working surface of the phase-type spatial light modulator 10 is only linearly polarized along the X-axis direction. At this time, the spectral distribution shown in formula (4) is expressed as:

[0061]

[0062] To achieve differential interferometry detection, the working surface of the phase-type spatial light modulator 10 is loaded with a desired phase modulation pattern, which is represented in spatial coordinates as follows:

[0063]

[0064] Where x0 and y0 represent the offset distances generated on the image acquisition module 13 along the X-axis and Y-axis directions, respectively, and are constants.

[0065] It should be noted that the phase range modulated by each pixel of the phase-type spatial light modulator 10 is between 0 and 2π. This means that the phase distribution finally loaded onto the phase-type spatial light modulator 10 must be between 0 and 2π to ensure accurate light field modulation. Considering that the phase modulation function of the light field is a periodic function with a fundamental period of 2π, the required phase distribution can be obtained by simply performing a phase wrapping process on the original phase distribution shown in formula (6). Therefore, the phase distribution actually loaded onto the phase-type spatial light modulator 10 is the result obtained after performing a phase wrapping process on the original phase distribution. According to the spatial Fourier transform of the lens, the spatial frequency (ξ,η) at the front focal plane of the second thin lens 11 has a relationship with the position coordinates (x,y). Therefore, the phase distribution shown in formula (6) can be expressed in frequency coordinates as follows:

[0066] θ(ξ,η)=2π(ξ·x0+η·y0) (7)

[0067] Therefore, the modulation function of the phase-type spatial light modulator 10 for the light field can be expressed as Mod(ξ,η)=e jθ(ξ,η) Where j represents the imaginary unit. Under the polarization modulation effect of the phase-type spatial light modulator 10, the spectral distribution shown in formula (5) is further expressed as:

[0068]

[0069] Finally, under the spatial Fourier transform effect of the second thin lens 11 and the polarization modulation effect of the second linear polarizer 12, a light field distribution with holographic differential interference effect is generated at the back focal plane of the second thin lens 11, which is expressed as:

[0070]

[0071] in, E c (x,y) is the ideal light field distribution produced by the object light wave s(x,y) after passing through a pairwise confocal system consisting of the microscope objective 3, the tube lens 4, the first thin lens 7, and the second thin lens 11, at the back focal plane of the second thin lens 11, i.e., the ideal magnified image of s(x,y). r (x,y) represents the complex amplitude point spread function of the confocal system composed of the first thin lens 7 and the second thin lens 11, s c (x-x0, y-y0) represents s c (x,y) is the function after translation along the vector (x0,y0).

[0072] Compared to E c (x,y), s c (x,y) still accurately carries the phase and amplitude information of sample 2, but their spatial resolution is limited. Therefore, s c (x,y)=A(x,y)·e jφ(x,y) Where A(x,y) and φ(x,y) are the sample amplitude information a(x,y) and sample phase information, respectively. The magnified image is obtained, but the spatial resolution of A(x,y) and φ(x,y) is limited. Therefore, obtaining the distribution of φ(x,y) is equivalent to obtaining the phase distribution of the sample. The light field distribution shown in Equation (9) is finally detected by the image acquisition module 13 located at the back focal plane of the second thin lens 11, and its intensity distribution is expressed as:

[0073]

[0074] Example 2

[0075] Based on Example 1, this example provides a rapid quantitative differential interferometry microscopy method based on holographic recording, including:

[0076] S1: The light intensity distribution of the sample is obtained using the rapid quantitative differential interference microscopy system described in Example 1. The expression for the intensity distribution is shown in Equation (10).

[0077] S2: Perform a spatial Fourier transform on the light intensity distribution to obtain the spectral distribution of the light intensity.

[0078] Step S2 in this embodiment specifically includes:

[0079] S2.1: Transform the expression for the light intensity distribution to obtain the transformed expression:

[0080]

[0081] Where * denotes the conjugate of the variables; B(x,y)=|s c (x,y)| 2 +|s c (x-x0, y-y0)| 2 ,

[0082] Performing a spatial Fourier transform on both sides of equation (11) yields I. c Spectral distribution of (x,y):

[0083]

[0084] S3: Based on the spectral distribution of the light intensity, the phase distribution of the sample is obtained using the holographic differential reconstruction method.

[0085] This step uses a method similar to digital holography to obtain e d The distribution of (x, y) is denoted here as the holographic differential reconstruction method. Specifically, it includes the following steps:

[0086] S3.1: The spectral distribution shown in formula (12) The whole body moves k in the opposite direction along the η coordinate axis. c The distance yields a new spectral distribution.

[0087] S3.2: Set a circular mask Mask(ξ,η) centered at the origin (0,0) of the spectral coordinates. This circular mask has a radius of... The value is 1 within the circular region and 0 in other regions. It should be noted that the radius is... The circular area is e d The effective range of the spectrum of (x,y).

[0088] S3.3: Multiply the spectral distribution by the circular mask Mask(ξ,η). get The distribution, and then the obtained Performing an inverse spatial Fourier transform on the distribution yields e. d The distribution of (x,y) is obtained, thus yielding the distribution of φ(x,y)-φ(x-x0,y-y0).

[0089] S3.4: Let y0 = 0 and in the phase modulation pattern shown in formula (6) At that time, the gradient distribution along the X-axis direction φ(x,y) is obtained:

[0090]

[0091] Similarly, let x0 = 0 and in the phase modulation pattern shown in formula (6) At that time, the gradient distribution along the Y-axis φ(x,y) is obtained:

[0092]

[0093] S3.5: Using the Frankot-Chellappa phase integration algorithm, the phase distribution of the sample under test can be quantitatively obtained.

[0094]

[0095] in, This represents the operation of Fourier transform in two-dimensional space. This represents the two-dimensional inverse space Fourier transform operation, and ε represents the regularization coefficient, which defaults to 0.0001.

[0096] Because the rapid quantitative differential interferometry microscopy system of this invention utilizes a phase-type spatial light modulator to precisely achieve shear shifting in any direction and to any degree, its imaging accuracy is extremely high, making it suitable for various types of applications. It should be noted that, in order for the rapid quantitative differential interferometry microscopy system proposed in this invention to achieve accurate quantitative phase imaging, the following requirements must be met between the various components:

[0097] (i) In order to process the spectral distributions of the 0th and ±1st order diffracted light differently at the confocal surface of the first thin lens 7 and the second thin lens 11, the spectral distributions of the 0th and ±1st order diffracted light must not overlap at the confocal surface of the first thin lens 7 and the second thin lens 11. Therefore, the following must be satisfied:

[0098] (ii) In order to filter and select the correct spectral distribution using a circular mask during the holographic differential reconstruction process, it is required that the three spectral distributions on the right side of formula (12) do not overlap with each other. Therefore, the following must be satisfied:

[0099] (III) In order to ensure that the working surface of the phase-type spatial light modulator 10 only covers the spectral distribution of the 0th and -1st order diffracted light at the confocal surface of the first thin lens 7 and the second thin lens 11, thereby achieving selective phase modulation, the following must be satisfied: and

[0100] (iv) In order to satisfy the sampling law and obtain a high-quality original image, the lateral spatial resolution of the system under vertical illumination must occupy at least four pixels of the image acquisition module 13. Therefore, the following must be satisfied:

[0101] (v) In order to satisfy the sampling law and achieve high-quality holographic recording of differential interference, the interference fringes must occupy at least four pixels of the image acquisition module 13. Therefore, the following must be satisfied:

[0102] (vi) In order to satisfy the sampling law during the holographic differential reconstruction process, that is, the signal with the highest frequency in the holographic differential interference intensity map is acquired by at least two pixels of the image acquisition module 13, the three spectral distributions on the right side of formula (12) must not exceed the edge of the spectral image. Therefore, the following must be satisfied:

[0103] The following simulation experiment further illustrates the effectiveness of the fast quantitative differential interferometry microscopy system based on holographic recording proposed in this invention.

[0104] In the simulation, the sample under test is set as a pure phase object, and its true phase distribution is as follows: Figure 3 As shown, and Figure 4 Give along Figure 3 The phase distribution of the horizontal white dashed line. Simultaneously, the transmission grating 5 is set to diffract along the Y-axis. To achieve holographic differential interference detection along the X-axis, the phase-type spatial light modulator 10 is loaded as follows... Figure 5 The phase distribution is shown. At this time, on the image acquisition module 13, the 0th order object wave undergoes shear shift along the X-axis relative to the -1st order object wave carrying the carrier frequency vector, and the holographic differential interference intensity map of the shear shift along the X-axis acquired by the image acquisition module 13 is shown in the figure. Figure 6 As shown.

[0105] right Figure 6 Performing a spatial Fourier transform yields the following: Figure 7The spectral distribution shown is then obtained using the holographic differential reconstruction method proposed in this invention, as shown below. Figure 8 The spectral distribution shown. For Figure 8 The spectral distribution shown is subjected to an inverse Fourier transform and phase extraction to obtain the phase difference distribution of the sample along the X-axis. Dividing this phase difference distribution by the shear offset along the X-axis yields the phase gradient distribution of the sample along the X-axis. Figure 9 As shown. Furthermore, to achieve holographic differential interference detection along the Y-axis, the phase-type spatial light modulator 10 is loaded as shown... Figure 10 The phase distribution is shown. At this time, on the image acquisition module 13, the 0th order object wave undergoes a shear shift along the Y-axis relative to the -1st order object wave carrying the carrier frequency vector, and the holographic differential interference intensity map of the shear shift along the Y-axis acquired by the image acquisition module 13 is shown in the figure. Figure 11 As shown. For Figure 11 Performing a spatial Fourier transform yields the following: Figure 12 The spectral distribution shown is then used to obtain the holographic differential reconstruction method proposed in this invention. Figure 13 The spectral distribution shown. For Figure 13 The spectral distribution shown is subjected to an inverse spatial Fourier transform and phase extraction to obtain the phase difference distribution of the sample along the Y-axis. Dividing this phase difference distribution by the shear offset along the Y-axis yields the phase gradient distribution of the sample along the Y-axis. Figure 14 As shown.

[0106] Finally, use formula (15) to... Figure 9 and Figure 14 Performing the Frankot-Chellappa phase integral yields the following result: Figure 15 The sample phase distribution is shown. Figure 16 Give along Figure 15 Phase distribution of the horizontal white dashed line. (Comparison) Figure 3 and Figure 15 and Figure 4 and Figure 16 As can be seen, the rapid quantitative differential interferometry microscopy system based on holographic recording proposed in this invention accurately reconstructs the phase distribution of the sample using only two original images acquired by the image acquisition module, demonstrating very high detection accuracy. It should be noted that, in the simulation process, this embodiment considers the reality that the system objective lens has a finite numerical aperture; therefore, Figure 15 and Figure 16 The phase distribution shown is relative to Figure 3 and Figure 4 The actual phase distributions shown all suffer from resolution degradation.

[0107] Subsequently, the rapid quantitative differential interferometry microscopy system of this embodiment was used to perform real rapid quantitative differential interferometry microscopy imaging on transparent live COS7 cells, and high-quality phase images were obtained, such as... Figures 17 to 19 As shown. First, a phase distribution with shear shift along the X-axis is applied to the phase-type spatial light modulator 10 to obtain the phase gradient distribution of the COS7 cells along the X-axis, as shown. Figure 17 As shown. Subsequently, a phase distribution with shear shift along the Y-axis is applied to the phase-type spatial light modulator 10 to obtain the phase gradient distribution of COS7 cells along the Y-axis, as shown. Figure 18 As shown. Finally, using formula (15) to... Figure 17 and Figure 18 Performing the Frankot-Chellappa phase integral on the phase gradient distribution shown, we obtain the following: Figure 19 The phase distribution of COS7 cells is shown. Specifically, Figure 20 Gives a comparison with Figure 19 Bright-field images of COS7 cells in the same field of view. (Comparison) Figure 19 and Figure 20 It can be seen that the rapid quantitative differential interferometry microscopy system based on holographic recording proposed in this invention can perform high-quality quantitative phase imaging of organelles such as mitochondria, lipid droplets, and black vacuoles in transparent living cells. It can capture the structural changes of the organelles themselves and the interactions between different organelles, which proves that the proposed device is feasible and effective.

[0108] In summary, the fast quantitative differential interferometry microscopy system based on holographic recording proposed in this invention achieves fast quantitative differential interferometry microscopy imaging by combining the polarization modulation characteristics of the grating carrier frequency and the phase-type spatial light modulator. This invention employs partially coherent light as illumination, avoiding speckle noise caused by highly coherent light sources. Therefore, this microscopy system exhibits high imaging quality and high spatial phase sensitivity. The invention utilizes a coaxial interference optical structure, exhibiting strong immunity to external disturbances and possessing high imaging stability and high temporal phase sensitivity. Compared to traditional quantitative differential interference microscopy, this invention utilizes a phase-type spatial light modulator to precisely achieve shear shifts in any direction and degree, resulting in extremely high imaging accuracy suitable for various applications. This invention utilizes a grating carrier frequency to achieve holographic recording of differential interference, enabling high-precision quantitative differential interference microscopy imaging of the sample using only two images. Therefore, this invention's rapid quantitative differential interference microscopy system possesses very high temporal and spatial resolution, along with very high imaging accuracy. Furthermore, this rapid quantitative differential interference microscopy system has a simple structure, is easy to assemble, and inherits the high axial resolution advantage of traditional differential interference phase-contrast microscopes, enabling three-dimensional quantitative phase imaging of thick tissue samples. Therefore, the proposed rapid quantitative differential interferometry microscopy system based on holographic recording can perform label-free, highly stable, highly sensitive and high-resolution in-situ quantitative phase imaging of the sample to be tested, and has very good scalability in terms of structure and function, and has great application value in the fields of biomedicine and industrial detection.

[0109] In the several embodiments provided by this invention, it should be understood that the apparatus and methods disclosed in this invention can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative. For example, the division of modules is merely a logical functional division, and in actual implementation, there may be other division methods. For example, multiple modules or components may be combined or integrated into another system, or some features may be ignored or not executed.

[0110] Furthermore, the functional modules in the various embodiments of the present invention can be integrated into one processing module, or each module can exist physically separately, or two or more modules can be integrated into one module. The integrated module can be implemented in hardware or in the form of hardware plus software functional modules.

[0111] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A rapid quantitative differential interferometry microscopy system based on holographic recording, characterized in that, The system includes a partially coherent illumination module (1) and, sequentially arranged along the optical axis of the partially coherent illumination module (1), a microscope objective (3), a tube lens (4), a transmission grating (5), a first linear polarizer (6), a first thin lens (7), a half-wave plate (8), a phase-type spatial light modulator (10), a second thin lens (11), a second linear polarizer (12), and an image acquisition module (13). The partially coherent illumination module (1) is used to generate partially coherent illumination light; the sample (2) is placed at the front focal plane of the microscope objective (3), and the transmission grating (5) is placed at the confocal plane of the tube lens (4) and the first thin lens (7); the first linear polarizer (6) is placed between the transmission grating (5) and the first thin lens (7); the polarization direction of the first linear polarizer (6) is along the Y-axis, and the first thin lens (7) only collects the 0th and ±1st order diffracted light carrying object light wave information and polarized along the Y-axis; The phase-type spatial light modulator (10) is disposed at the confocal surface of the first thin lens (7) and the second thin lens (11), and the working surface of the phase-type spatial light modulator (10) only covers the spectral distribution of the 0th and -1st order diffracted light. The half-wave plate (8) is disposed between the first thin lens (7) and the phase-type spatial light modulator (10) and is adjacent to the working surface of the phase-type spatial light modulator (10), which enables the spectral distribution of the -1st order diffracted light reaching the working surface of the phase-type spatial light modulator (10) to be linearly polarized along the X-axis direction. The X-axis direction and the Y-axis direction are perpendicular to each other and both are perpendicular to the optical axis direction of the partially coherent illumination module (1). The phase-type spatial light modulator (10) can perform phase modulation on the spectral distribution of the 0th order diffracted light; the second linear polarizer (12) is disposed between the second thin lens (11) and the image acquisition module (13) for polarization adjustment of the 0th order diffracted light and the -1st order diffracted light with the same polarization direction; the image acquisition module (13) is used for image acquisition and recording. The phase modulation pattern loaded on the working surface of the phase-type spatial light modulator (10) is represented by spatial coordinates as follows: in, These represent the offset distances generated along the X-axis and Y-axis directions on the image acquisition module (13), respectively. This indicates the center wavelength of the emitted light from the partially coherent illumination module (1). f 2 represents the focal length of the second thin lens (11); The parameters of the microscope objective (3), the tube lens (4), and the transmission grating (5) satisfy: ， in, This indicates the center wavelength of the emitted light from the partially coherent illumination module (1). f obj and NA These represent the focal length and numerical aperture of the microscope objective (3), respectively. This indicates the focal length of the tube lens (4). d This represents the grating constant of the transmission grating (5); The expression for the light intensity distribution detected by the image acquisition module (13) is: ， in, j Represents the imaginary unit. , This represents the convolution operation. The complex amplitude point spread function represents the confocal system composed of the microscope objective (3) and the tube lens (4). The complex amplitude point spread function represents the confocal system composed of the first thin lens (7) and the second thin lens (11); object light waves The ideal light field distribution generated at the back focal plane of the second thin lens (11) , Represents horizontal spatial coordinates. This indicates the amplitude information of the sample. Indicates the phase information of the sample; express along The function after vector translation d This represents the grating constant of the transmission grating (5). f 1 represents the focal length of the first thin lens (7). f 2 represents the focal length of the second thin lens (11).

2. The rapid quantitative differential interferometry microscopy system based on holographic recording according to claim 1, characterized in that, It also includes a light-shielding plate (9) disposed at the confocal surface of the first thin lens (7) and the second thin lens (11), the light-shielding plate (9) being used to block the spectral distribution of the +1 order diffracted light.

3. The rapid quantitative differential interferometry microscopy system based on holographic recording according to claim 2, characterized in that, The width of the working surface of the phase-type spatial light modulator (10) SLM_width and length SLM_height satisfy: ， ， in, f 1 represents the focal length of the first thin lens (7).

4. The rapid quantitative differential interferometry microscopy system based on holographic recording according to claim 3, characterized in that, The pixel size of the image acquisition module (13) satisfies: ， ， ， in, f 2 represents the focal length of the second thin lens (11).

5. A rapid quantitative differential interferometry microscopy method based on holographic recording, characterized in that, include: S1: Obtain the light intensity distribution of the sample using the rapid quantitative differential interferometry microscopy system according to any one of claims 1 to 4; S2: Perform a spatial Fourier transform on the light intensity distribution to obtain the spectral distribution of the light intensity; S3: Based on the spectral distribution of the light intensity, the phase distribution of the sample is obtained using the holographic differential reconstruction method.

6. The rapid quantitative differential interferometry microscopy method based on holographic recording according to claim 5, characterized in that, S2 includes: S2.1: Transform the expression for the light intensity distribution to obtain the transformed expression: in, Indicates conjugate. , , , and These represent sample amplitude information. and sample phase information Magnified image; S2.2: Perform a spatial Fourier transform on both sides of the transformed expression simultaneously to obtain the spectral distribution of the light intensity: in, Representation of spatial coordinates The corresponding spectral coordinates, where ~ represents the two-dimensional Fourier transform of the corresponding variable.

7. The rapid quantitative differential interferometry microscopy method based on holographic recording according to claim 6, characterized in that, S3 includes: S3.1: Distribute the spectrum Overall along Shift in the negative direction of the coordinate axis Distance yields a new spectral distribution. ; S3.2: Set a circular mask centered on the origin of the spectral coordinates. The circular mask has a radius of The value is 1 within the circular region and 0 in other regions; S3.3: Using the circular mask Multiplied by the new spectral distribution get The distribution of the obtained Performing an inverse spatial Fourier transform on the distribution yields... The distribution, thereby obtaining Distribution; S3.4: In the phase modulation pattern loaded on the phase-type spatial light modulator (10), =0 and ,get Gradient distribution: ， In the phase modulation pattern loaded by the phase-type spatial light modulator (10), =0 and At that time, Gradient distribution: ； S3.5: Quantitatively obtain the phase distribution of the sample to be tested: in, This represents the operation of Fourier transform in two-dimensional space. This represents the two-dimensional inverse Fourier transform operation. This represents the regularization coefficient.

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