Large numerical aperture quantitative phase microscopy system and method based on KK relation

Abstract

The invention discloses a large numerical aperture quantitative phase microscopy system and method based on a KK relation. The system comprises an illumination module, a microscope objective, a lens cone lens, a visual field limiting module, a first thin lens, a linear polarizer, a second thin lens, a first phase type spatial light modulator, a third thin lens, a second phase type spatial light modulator, a fourth thin lens and a polarization camera which are arranged in sequence, and the illumination module performs transmission type oblique illumination on a sample; the visual field limiting module is used for carrying out space limitation on an illumination field and a scattering field; the linear polarizer is used for decomposing a light field into an X polarization component and a Y polarization component; the first phase-type spatial light modulator is used for modulating the Y polarization illumination field and the Y polarization scattering field; the second phase-type spatial light modulator is used for performing phase modulation on the Y polarization scattered field; the polarization camera is used for obtaining intensity distribution and hologram distribution of the illumination field. According to the invention, the sample can be subjected to label-free, high-resolution and high-stability in-situ quantitative detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of optical microscopic imaging, and particularly relates to a large numerical aperture quantitative phase microscopic system and method based on a KK relationship. BACKGROUND

[0002] Quantitative phase microscopy (QPM) as a new label-free microscopic technology plays an important role in the fields of biomedical research, industrial detection and material science. QPM combines phase imaging and optical microscopy, and can realize high-resolution and high-contrast imaging of transparent samples without fluorescent labeling. In the field of life medical research, QPM can realize high-resolution imaging of organelles in living cells in a natural state, so as to explore the mechanism of life phenomena. In the field of industrial detection, QPM can non-destructively detect the surface morphology and internal defects of micro-nano devices, so as to characterize the machining precision.

[0003] Digital holographic microscopy as a pioneer of QPM has obtained strong development in recent years and plays a key role in many fields. However, digital holographic microscopy still has certain limitations, and it cannot simultaneously ensure imaging quality, spatial resolution, imaging stability and spatial bandwidth product. In order to improve the comprehensive imaging ability of QPM, researchers have proposed a series of new QPM technologies in recent years, such as quantitative differential interference microscopy, quantitative phase contrast microscopy, QPM based on Gerchberg-Saxton (GS) iteration, QPM based on asymmetric illumination and deconvolution, QPM based on light intensity transport equation, Fourier ptychographic microscopy based on iterative operation, etc., collectively referred to as on-axis QPM. On-axis QPM uses partially coherent light as the illumination light source, and some of the technologies are based on common-path interference optical structures to realize quantitative phase imaging, without the need for additional reference light. Another part of the technology is based on the diffraction propagation effect of light field in space to realize quantitative phase imaging. Therefore, on-axis QPM has the advantages of simple structure, high stability, high imaging quality, high spatial resolution, low cost and full use of the spatial bandwidth product of the imaging system. On-axis QPM has a wider application prospect. However, on-axis QPM still has some limitations in practical application, such as the need to collect multiple intensity images under different angles of oblique illumination, the need to accurately move the sample along the axial direction multiple times, the need to calibrate the imaging plane in the transverse or axial direction, the need to perform complex iterative calculations, and the need to approximately model the imaging system.

[0004] In recent years, researchers have proposed a holographic imaging technique based on the Kramers-Kronig relationship (KK relationship), which greatly improves the space-bandwidth product of off-axis digital holographic microscopy. However, the optical structure of off-axis interference makes the KK relationship-based holographic imaging technique extremely susceptible to external interference. Subsequently, researchers introduced the KK relationship into on-axis quantitative phase microscopy, realizing KK relationship-based on-axis quantitative phase microscopy, which shows great potential in the application of quantitative phase microscopy imaging. The KK relationship-based on-axis quantitative phase microscopy has all the advantages of on-axis quantitative phase microscopy, i.e., simple structure, high stability, high imaging quality, high spatial resolution, low cost, and high space-bandwidth product. In KK relationship-based on-axis quantitative phase microscopy, only a single intensity image is needed to obtain the object light field distribution generated by the sample under oblique illumination at a certain angle through Hilbert transform. Therefore, the KK relationship-based on-axis quantitative phase microscopy compensates for many existing defects of on-axis quantitative phase microscopy, which does not require complex iterative operations, does not require the collection of multiple intensity images under oblique illumination at a certain angle, does not require the accurate movement of the sample along the axial direction multiple times, does not require the lateral or axial alignment of the imaging plane, and does not require the approximate modeling of the imaging system. However, the KK relationship-based on-axis quantitative phase microscopy requires that the transverse wave vector frequency of all oblique illuminations must be consistent with the cutoff frequency of the objective lens. This is extremely easy to achieve for low numerical aperture air objectives, but it is extremely difficult to achieve for high numerical aperture objectives such as oil immersion objectives. Therefore, the KK relationship-based on-axis quantitative phase microscopy cannot be applied to high numerical aperture objectives at present, and its spatial resolution is still insufficient to study fine structures such as subcellular organelles in living cells. SUMMARY

[0005] To solve the above problems in the prior art, the present application provides a KK relationship-based high numerical aperture quantitative phase microscopy system and method. The technical problem to be solved by the present application is solved by the following technical scheme: One aspect of the present application provides a KK relationship-based high numerical aperture quantitative phase microscopy system, comprising, in sequence, an illumination module, a microscope objective, a lens barrel lens, a field-of-view limiting module, a first thin lens, a linear polarizer, a second thin lens, a first phase-type spatial light modulator, a third thin lens, a second phase-type spatial light modulator, a fourth thin lens, and a polarization camera, wherein A sample is arranged at the front focal plane of the microscope objective, the illumination module can emit a non-polarized and partially coherent quasi-plane wave for transmissive oblique illumination of the sample to generate an object light field, the object light field includes an illumination field unaffected by the sample and a scattering field carrying sample information; The microscope objective and the lens barrel lens have a confocal surface, the field-of-view limiting module is arranged at the confocal surface of the lens barrel lens and the first thin lens, and the field-of-view limiting module is used for spatially limiting the illumination field and the scattering field from the lens barrel lens to obtain a distribution-limited illumination field and a distribution-limited scattering field. The first thin lens and the second thin lens constitute a confocal system, and the linear polarizer is used for decomposing the distribution-limited illumination field and the distribution-limited scattering field into two mutually orthogonal X-polarization components and Y-polarization components, wherein the X-polarization components include X-polarization illumination fields and X-polarization scattering fields, and the Y-polarization components include Y-polarization illumination fields and Y-polarization scattering fields. The first phase-type spatial light modulator is arranged at the back focal surface of the second thin lens, is loaded with a pre-designed first phase modulation pattern, and is used for modulating the Y-polarization illumination fields and the Y-polarization scattering fields. The front focal surface of the third thin lens coincides with the working surface of the first phase-type spatial light modulator, the second phase-type spatial light modulator is arranged at the back focal surface of the third thin lens, is loaded with a pre-designed second phase modulation pattern, and is used for phase-modulating the Y-polarization scattering fields. The front focal surface of the fourth thin lens coincides with the working surface of the second phase-type spatial light modulator, and the polarization camera is arranged at the back focal surface of the fourth thin lens to obtain the intensity distribution of the illumination field of the sample and the hologram distribution under the illumination of the illumination module.

[0006] Another aspect of the present application provides a large numerical aperture quantitative phase microscopy method based on a KK relationship, comprising: S1: obtaining the intensity distribution of the illumination field and the hologram distribution of the sample generated under the oblique illumination of different light-emitting diodes by using the large numerical aperture quantitative phase microscopy based on the KK relationship, and quantitatively obtaining the X-polarization object field distribution of the sample generated under the oblique illumination of different light-emitting diodes by using the KK relationship; S2: obtaining a two-dimensional quantitative phase image of the sample or a three-dimensional refractive index distribution of the sample according to the X-polarization object field distribution of the sample generated under the oblique illumination of different light-emitting diodes.

[0007] Compared with the prior art, the present application has the following beneficial effects: 1. The application provides a large numerical aperture quantitative phase microscopy system and method based on KK relation, which has the following advantages: first, the phase modulation of polarization is implemented on the object light field and its spectrum by two phase space light modulators, which effectively solves the problem that the transverse wave frequency of the illumination field must be strictly consistent with the cutoff frequency of the detection objective in the coaxial quantitative phase microscopy imaging based on KK relation, so the application is suitable for all types of objective; the system of the application does not need an additional illumination objective to be coupled with the detection objective, and only needs the annularly distributed light-emitting diode on the illumination module to directly perform partial coherent illumination on the sample, so that the structure of the imaging system is greatly simplified, and the application has the advantages of simple structure, high imaging quality, high spatial resolution, low cost and high space-bandwidth product; second, the optical structure of common-path interference makes the microscopic system of the application have very strong immunity to external disturbance and very high stability; third, the application does not need complicated iterative operation, and only needs Hilbert transform and single exposure to obtain the object light field distribution of the sample under the oblique illumination at a certain angle, so the microscopic system of the application has very high time resolution and imaging robustness; fourth, the application can perform label-free and high-resolution quantitative phase imaging on fine structures such as subcellular organelles in living cells; in addition, the application can be coupled with any microscopic imaging technology to realize multi-mode imaging; finally, the application can not only realize two-dimensional fast quantitative phase imaging of the sample, but also can perform three-dimensional refractive index imaging of the sample.

[0008] 2. The large numerical aperture quantitative phase microscopy system and method based on KK relation can perform label-free, high-resolution, high-stability and high-speed in-situ quantitative detection on the sample, and has very good expandability in structure and function, and has great application value in the fields of life science research and industrial detection.

[0009] The application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a light path schematic diagram of a large numerical aperture quantitative phase microscopy system based on KK relation provided by the application; Figure 2 is a polarizing direction schematic diagram of a linear polarizer; Figure 3 is a schematic diagram of the distribution of X polarization spectrum and Y polarization spectrum at the back focal plane of the third thin lens; Figure 4 is a schematic diagram of the light field distribution at the back focal plane of the fourth thin lens; Figure 5Fig. 1 is a schematic diagram of a spectral distribution of an intensity pattern produced by coherent superposition of a Y-polarized illumination field and an X-polarized object field in a 45-degree polarization direction under oblique illumination at a certain angle; Figure 6 Fig. 4 is a two-dimensional quantitative phase image of a living COS7 cell obtained by the large numerical aperture quantitative phase microscopy system based on the KK relationship.

[0011] Reference signs: 1-illumination module; 2-sample; 3-microscopic objective; 4-lens barrel lens; 5-field limitation module; 6-first thin lens; 7-linear polarizer; 8-second thin lens; 9-first non-polarization beam splitter prism; 10-first phase-type spatial light modulator; 11-third thin lens; 12-second non-polarization beam splitter prism; 13-second phase-type spatial light modulator; 14-fourth thin lens; 15-polarization camera. DETAILED DESCRIPTION

[0012] In order to further illustrate the technical means and effects taken by the present application to achieve the predetermined purposes, the large numerical aperture quantitative phase microscopy system and method based on the KK relationship according to the present application are described in detail below in combination with the accompanying drawings and specific embodiments.

[0013] The foregoing and other technical contents, features and effects of the present application can be clearly presented in the following detailed description of specific embodiments in combination with the accompanying drawings. Through the description of specific embodiments, the technical means and effects taken by the present application to achieve the predetermined purposes can be understood more deeply and specifically. However, the accompanying drawings are provided for reference and illustration only, and are not intended to limit the technical solutions of the present application.

[0014] It should be noted that, in this document, relational terms such as first and second, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without more limitations, an element defined by an utterance "comprising a" does not exclude the existence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0015] Embodiment one Please refer to Figure 1 , Figure 1The KK relationship-based large numerical aperture quantitative phase microscopy system is provided by the embodiment of the present application. The large numerical aperture quantitative phase microscopy system comprises an illumination module 1, a microscope objective 3, a lens barrel lens 4, a field of view limiting module 5, a first thin lens 6, a linear polarizer 7, a second thin lens 8, a first phase-type spatial light modulator 10, a third thin lens 11, a second phase-type spatial light modulator 13, a fourth thin lens 14 and a polarization camera 15 arranged in sequence, wherein the sample 2 is arranged at the front focal plane of the microscope objective 3, the illumination module 1 can emit a non-polarized and partially coherent quasi-plane wave, which is used for transmission oblique illumination of the sample 2 to generate an object light field, the object light field comprises an illumination field not affected by the sample and a scattering field carrying sample information; the microscope objective 3 and the lens barrel lens 4 have a confocal plane, the field of view limiting module 5 is arranged at the confocal plane of the lens barrel lens 4 and the first thin lens 6, and the field of view limiting module 5 is used for spatially limiting the illumination field and the scattering field from the lens barrel lens 4 to obtain a distribution-limited illumination field and a distribution-limited scattering field; the first thin lens 6 and the second thin lens 8 form a confocal system, and the linear polarizer 7 is used for decomposing the distribution-limited illumination field and the distribution-limited scattering field into two mutually orthogonal X-polarization components and Y-polarization components, wherein the X-polarization components comprise X-polarization illumination fields and X-polarization scattering fields, and the Y-polarization components comprise Y-polarization illumination fields and Y-polarization scattering fields; the first phase-type spatial light modulator is arranged at the back focal plane of the second thin lens 8, loaded with a pre-designed first phase modulation pattern, and used for modulating the Y-polarization illumination fields and the Y-polarization scattering fields; the front focal plane of the third thin lens 11 coincides with the working surface of the first phase-type spatial light modulator 10, the second phase-type spatial light modulator 13 is arranged at the back focal plane of the third thin lens 11, loaded with a pre-designed second phase modulation pattern, and used for phase modulation of the Y-polarization scattering fields; the front focal plane of the fourth thin lens 14 coincides with the working surface of the second phase-type spatial light modulator 13, and the polarization camera 15 is arranged at the back focal plane of the fourth thin lens 14 to obtain the intensity distribution of the illumination field of the sample and the hologram distribution under the illumination of the illumination module 1. It should be noted that in the KK relationship-based large numerical aperture quantitative phase microscopy system, the geometric centers or axes of all devices coincide with the optical axis of the system.

[0016] The large numerical aperture quantitative phase microscopy system further comprises a first non-polarizing beam splitter prism 9 and a second non-polarizing beam splitter prism 12, wherein the first non-polarizing beam splitter prism 9 is arranged between the second thin lens 8 and the first phase-type spatial light modulator 10, used for transmitting the light beam from the second thin lens 8 to the first phase-type spatial light modulator 10, and reflecting the light beam from the first phase-type spatial light modulator 10 to the third thin lens 11; the second non-polarizing beam splitter prism 12 is arranged between the third thin lens 11 and the second phase-type spatial light modulator 13, used for transmitting the light beam from the third thin lens 11 to the second phase-type spatial light modulator 13, and reflecting the light beam from the second phase-type spatial light modulator 13 to the fourth thin lens 14.

[0017] The illumination module 1 of the embodiment comprises a plurality of ring-shaped uniformly distributed light-emitting diodes, which can be sequentially turned on, and only one light-emitting diode is turned on at the same time; when one light-emitting diode is turned on, a quasi-plane wave which is unpolarized and partially coherent can be emitted.

[0018] The plurality of light-emitting diodes are completely identical, and are designed to perform transmission oblique illumination on a sample. In the process of quantitatively detecting the sample, the light-emitting diodes on the illumination module 1 are sequentially turned on, and when a certain light-emitting diode is turned on, a quasi-plane wave which is unpolarized and partially coherent is emitted, and transmission oblique illumination is performed on the sample 2 located at the front focal plane of the microscope objective 3. In the embodiment, the sample 2 is weakly scattering. Since the present application belongs to the field of microscopic imaging, the effective field of view range of detection is in the order of several hundred microns, which can be almost ignored relative to the size of the light-emitting diode on the illumination module 1. Therefore, for the sample 2, the quasi-plane wave generated by the light-emitting diode on the illumination module 1 can be approximated as a plane wave.

[0019] After the sample 2 interacts with the tilted plane wave, a certain distribution of object light field is generated in space, which is composed of an illumination field not affected by the sample and a scattering field carrying sample information. After being magnified and imaged by the confocal system composed of the microscope objective 3 and the tube lens 4, the illumination field and the scattering field at the front focal plane of the microscope objective 3 are magnified and propagated to the confocal plane of the tube lens 4 and the first thin lens 6. During this process, the illumination field and the scattering field successively undergo spatial Fourier transform by the microscope objective 3 and the tube lens 4, and their frequency spectra are limited by the pupil aperture of the microscope objective 3. Therefore, the scattering field propagated to the confocal plane of the tube lens 4 and the first thin lens 6 has a limited frequency spectrum range.

[0020] At the same time, a field of view limiting module 5 is positioned at the confocal plane between the tube lens 4 and the first thin lens 6. This field of view limiting module 5 comprises a light-transmitting region and a light-blocking region. The light-transmitting region, located at the center of the field of view limiting module 5, does not modulate the light field and is square in shape. The geometric center of the light-transmitting region coincides with the system optical axis. The light-blocking region surrounds the light-transmitting region and spatially limits the illumination and scattered fields from the tube lens 4, resulting in distribution-restricted illumination and scattered fields. Therefore, after being restricted by the field of view limiting module 5, the illumination and scattered fields propagating to the confocal plane between the tube lens 4 and the first thin lens 6 have a limited spatial range (i.e., the range of the light-transmitting region), collectively referred to herein as the distribution-restricted illumination and scattered fields. After being imaged by the confocal system formed by the first thin lens 6 and the second thin lens 8, the distribution-restricted illumination and scattered fields are propagated to the back focal plane of the second thin lens 8. During this process, the distribution-restricted illumination and scattered fields undergo the spatial Fourier transform effects of the first and second thin lenses 6 and 8, and the modulation effect of the linear polarizer 7.

[0021] The linear polarizing plate 7 of this embodiment is arranged near the second thin lens 8. The polarization direction of the linear polarizing plate 7 is perpendicular to the Z axis, and the angle between the polarization direction of the linear polarizing plate 7 and the positive direction of the X axis is greater than the angle between the polarization direction of the linear polarizing plate 7 and the positive direction of the Y axis. Figure 2 As shown, the Z axis coincides with the system optical axis, and the positive direction of the Z axis is along the light propagation direction; the X axis and the Y axis are perpendicular to each other and perpendicular to the Z axis; the linear polarizer 7 is used to decompose the distribution-restricted illumination field and scattered field into an illumination field and scattered field linearly polarized along the X axis and an illumination field and scattered field linearly polarized along the Y axis. Therefore, the distribution-restricted illumination field and scattered field propagated to the back focal plane of the second thin lens 8 can be decomposed into two mutually orthogonal polarization components, namely, an illumination field and scattered field linearly polarized along the X axis (referred to as the X-polarized illumination field and the X-polarized scattered field) and an illumination field and scattered field linearly polarized along the Y axis (referred to as the Y-polarized illumination field and the Y-polarized scattered field). It should be noted that the X-polarized illumination field and the X-polarized scattered field, as well as the Y-polarized illumination field and the Y-polarized scattered field mentioned below are all distribution-restricted, but this will not be specifically stated.

[0022] Subsequently, the light field from the second thin lens 8 is split by the first non-polarization beam splitter prism 9 , with 50% of the energy being reflected away from the system and 50% of the energy being transmitted to the first phase-type spatial light modulator 10 .

[0023] The working surface of the first phase-type spatial light modulator 10 completely covers the distribution-limited illumination field and the scattering field. The first phase-type spatial light modulator 10 is loaded with a pre-designed first phase modulation pattern, which has polarization selectivity in the modulation of the light field, and only acts on the light field linearly polarized along the Y-axis direction, but does not act on the light field linearly polarized along the X-axis direction. Therefore, for the X-polarized illumination field and the X-polarized scattering field, the first phase-type spatial light modulator 10 only acts as a mirror reflection. Specifically, under the mirror reflection of the first phase-type spatial light modulator 10, the X-polarized illumination field and the X-polarized scattering field enter the first non-polarizing beam splitter prism 9, of which only 50% of the energy is reflected into the third thin lens 11.

[0024] Since the front focal surface of the third thin lens 11 coincides with the working surface of the first phase-type spatial light modulator 10. Therefore, the X-polarized illumination field and the X-polarized scattering field propagating into the third thin lens 11 reflected by the first non-polarizing beam splitter prism 9 actually experience the spatial Fourier transform of the third thin lens 11. Therefore, the spectral information of the X-polarized illumination field and the X-polarized scattering field (collectively referred to as X-polarized spectrum) is presented at the back focal surface of the third thin lens 11. The X-polarized spectrum is presented as a circular region (referred to as X-polarized circular region), and its center of circle coincides with the optical axis of the system (the back focal point of the third thin lens 11). Since the transmission oblique illumination of the light-emitting diode on the illumination module 1 to the sample 2 is a tilted plane wave. Therefore, at the back focal surface of the third thin lens 11, the spectral information of the X-polarized illumination field is presented as a focal point deviated from the optical axis of the system, and the spectral information of the X-polarized scattering field is distributed in a circular region concentric with the back focal point of the third thin lens 11 (X-polarized circular region), as shown in FIG. 4. Figure 3 Since the quantitative phase microscopy system of the present application does not require the transverse frequency of the illumination wave vector to be strictly consistent with the cutoff frequency of the detection objective, at the back focal surface of the third thin lens 11, the spectrum of the X-polarized illumination field (focal point) is located in the X-polarized circular region, and far away from its edge, as shown in FIG. 4. Figure 3

[0025] At the same time, the Y-polarized illumination field and the Y-polarized scattering field are modulated by the first phase-type spatial light modulator 10, and experience the beam splitting of the first non-polarizing beam splitter prism 9 and the spatial Fourier transform of the third thin lens 11. Therefore, the spectral information of the Y-polarized illumination field and the Y-polarized scattering field after phase modulation (collectively referred to as Y-polarized spectrum) is presented at the back focal surface of the third thin lens 11. The Y-polarized spectrum is presented as a circular region (referred to as Y-polarized circular region), and its center of circle deviates from the optical axis of the system (the back focal point of the third thin lens 11), as shown in FIG. 5. Figure 3 ​The Y-polarization spectrum and the X-polarization spectrum contain exactly the same information, but the former is laterally shifted with respect to the latter. Specifically, the spectrum (focal point) of the Y-polarized illumination field is located at the edge of the X-polarization circle. Moreover, the spectrum (focal point) of the Y-polarized illumination field, the spectrum (focal point) of the X-polarized illumination field, and the center of the X-polarization circle are located on a straight line, as shown in FIG. 2B. Figure 3 It is noted that, due to the light-splitting effect of the second non-polarizing light-splitting prism 12, only 50% of the X-polarization spectrum and the Y-polarization spectrum reach the back focal plane of the third thin lens 11.

[0026] Meanwhile, a second phase-type spatial light modulator 13 is disposed at the back focal plane of the third thin lens 11, and is loaded with a pre-designed second phase modulation pattern. The second phase-type spatial light modulator 13 has polarization selectivity in modulation of the light field, and only acts on the light field linearly polarized along the Y-axis direction, but does not act on the light field linearly polarized along the X-axis direction. Therefore, at the back focal plane of the third thin lens 11, only the Y-polarization spectrum is subjected to the modulation effect of the second phase-type spatial light modulator 13, and the X-polarization spectrum is only subjected to the mirror reflection effect of the second phase-type spatial light modulator 13. The second phase modulation pattern loaded by the second phase-type spatial light modulator 13 is divided into two regions: a circular region covering the spectrum of the Y-polarized illumination field and other regions. The phase values loaded by the circular region covering the spectrum of the Y-polarized illumination field are all 0, and the radius of the circular region is determined by the size of the light-emitting diode and the system parameters; and the other regions are loaded with a pre-designed phase modulation pattern. Therefore, for the spectrum of the Y-polarized illumination field and the X-polarization spectrum, the second phase-type spatial light modulator 13 only plays a mirror reflection role; and for the spectrum of the Y-polarized scattering field, the second phase-type spatial light modulator 13 performs predetermined phase modulation. Subsequently, after the light-splitting effect (50% of the energy is reflected into the subsequent system) of the second non-polarizing light-splitting prism 12 and the spatial Fourier transform effect of the fourth thin lens 14, the X-polarized illumination field, the X-polarized scattering field, the Y-polarized illumination field, and the Y-polarized scattering field propagate to the back focal plane of the fourth thin lens 14. Among them, the X-polarized illumination field, the X-polarized scattering field, and the Y-polarized illumination field are completely coincided together. However, the Y-polarized scattering field is far away from the Y-polarized illumination field, the X-polarized illumination field, and the X-polarized scattering field, and does not overlap with them, as shown in FIG. 2C. Figure 4

[0027] ​Meanwhile, a polarization camera 15 is arranged at the back focal plane of the fourth thin lens 14, and the effective working plane of the polarization camera 15 only covers the Y-polarized illumination field, the X-polarized illumination field and the X-polarized scattering field, but does not cover the Y-polarized scattering field. Therefore, at the back focal plane of the fourth thin lens 14, the Y-polarized scattering field is equivalent to non-existence. Based on this, the light field actually collected by the polarization camera 15 is composed of two parts: the Y-polarized illumination field and the X-polarized object light field (the sum of the X-polarized illumination field and the X-polarized scattering field). As described above, at the confocal plane of the third thin lens 11 and the fourth thin lens 14, the spectrum (focal point) of the Y-polarized illumination field is located at the edge position of the X-polarized circle, which means that the transverse wave vector frequency of the Y-polarized illumination field is exactly equal to the cutoff frequency of the X-polarized object light field; in addition, since the angle between the polarization direction of the linear polarizer 7 and the positive direction of the X-axis (vertically upward in the paper) is larger than the angle between the polarization direction of the linear polarizer 7 and the positive direction of the Y-axis (outward perpendicular to the paper), and for a weak scattering sample, the energy of the illumination field is much larger than the energy of the scattering field, therefore, at the back focal plane of the fourth thin lens 14, the energy of the Y-polarized illumination field is greater than the energy of the X-polarized object light field.

[0028] Based on this, the quantitative phase microscopy system proposed in the present application meets the condition for implementing holographic imaging based on the KK relationship. Therefore, the object light field distribution generated by the sample 2 under the oblique illumination of a certain light-emitting diode can be obtained through the KK relationship. It is worth noting that a single exposure of the polarization camera 15 can simultaneously provide the light intensity distributions of four polarization directions, i.e. 0 degree (X-axis), 45 degrees, 90 degrees (Y-axis) and 135 degrees. Among them, the light intensity distribution of the 90-degree direction is the light intensity distribution of the Y-polarized illumination field, and the intensity distribution of the 45-degree direction is the hologram distribution after the coherent superposition of the Y-polarized illumination field and the X-polarized object light field in the direction simultaneously forming 45 degrees with the positive direction of the X-axis and the positive direction of the Y-axis. Therefore, a single exposure of the polarization camera 15 can simultaneously provide all the intensity distributions required for holographic imaging based on the KK relationship, i.e. the light intensity distribution of the illumination field and the hologram distribution.

[0029] Then, all the light-emitting diodes on the illumination module 1 are switched in turn, and the above operation is repeated, so that the object light field distributions generated by the sample (2) under the oblique illumination of different light-emitting diodes can be obtained. Among them, the first phase modulation pattern loaded on the first phase-type spatial light modulator 10 is related to the position of the light-emitting diode to be lit and the cutoff frequency of the system; and the second phase modulation pattern loaded on the second phase-type spatial light modulator 13 is related to the position of the light-emitting diode to be lit, the first phase modulation pattern loaded on the first phase-type spatial light modulator 10 and the effective working range of the polarization camera 15, and the expressions of the first phase modulation pattern and the second phase modulation pattern will be described in the following continuation. Finally, by subsequent processing of all the obtained object light field distributions, two-dimensional quantitative phase imaging or three-dimensional refractive index imaging can be realized.

[0030] In summary, the large numerical aperture quantitative phase microscopy system based on KK relation can effectively solve the problem that the transverse wave vector frequency of the illumination field must be strictly consistent with the cutoff frequency of the detection objective lens in the coaxial quantitative phase microscopy imaging based on KK relation, and is suitable for all types of objective lenses. The imaging system of the present application directly illuminates the sample with a light-emitting diode, which has a simple structure, high imaging quality and high spatial resolution. The present application adopts a common path interference optical structure, which has very high stability. The present application does not need to perform complex iterative operation, and only needs to obtain the object light field distribution generated by the sample under different oblique illumination through Hilbert transform, so the imaging system of the present application has very high time resolution, imaging robustness and spatial bandwidth product. The imaging system of the present application can perform label-free, high-resolution, two-dimensional fast quantitative phase imaging and three-dimensional refractive index imaging of subcellular organelles in living cells. Therefore, the quantitative phase microscopy instrument based on KK relation and suitable for large numerical aperture objective lens can perform label-free, high-resolution, high-stability and high-speed in-situ quantitative detection of the sample, and has very good expandability in structure and function, and has great application value in the fields of life science research and industrial detection.

[0031] Example two On the basis of example one, the detailed imaging mechanism of the large numerical aperture quantitative phase microscopy system based on KK relation is as follows.

[0032] Firstly, the OXYZ three-dimensional space coordinate system is established with the object focal point of the microscopic objective lens 3 as the origin, wherein the vertical upward in the paper is the positive direction of the X axis, the outward perpendicular to the paper is the positive direction of the Y axis, and the horizontal right in the paper is the positive direction of the Z axis (the Z axis coincides with the system optical axis), as shown in FIG. 1. Figure 1 It should be noted that in the subsequent optical path, although the reflection of the phase type spatial light modulator changes the propagation of the optical path, the relative relationship of the coordinate system will not change, that is, the Z axis always coincides with the system optical axis, and the positive direction of the Y axis always points outward perpendicular to the paper. At the same time, let the focal length and numerical aperture of the microscopic objective lens 3 be and NA ; the focal length of the lens barrel lens 4 is ; the focal length of the first thin lens 6 is f 1; the focal length of the second thin lens 8 is f 2; the focal length of the third thin lens 11 is f 3; the focal length of the fourth thin lens 14 is f 4; the light transmission region of the field of view limiting module 5 is set as a square with a side length of b .

[0033] In the present embodiment, sample 2 is weakly scattering, which has little effect on the amplitude information of the illumination light. Sample 2 is placed at the front focal plane of microscope objective 3, and its modulating effect on the transmitted light can be represented by a two-dimensional function , where represents the transverse spatial coordinates, represents the phase information of sample 2, j represents the imaginary unit. When a certain light-emitting diode on illumination module 1 is turned on, it emits a non-polarized and partially coherent plane wave (center wavelength ), and performs transmission oblique illumination on sample 2. The oblique illumination is represented as , where m represents the serial number of the light-emitting diode; represents the three-dimensional spatial coordinates; represents the wave number in vacuum; represents the refractive index of the medium matched with microscope objective 3; represents the unit vector along the wave vector direction of the illumination light, here, represents the included angle between the wave vector of the illumination light and the positive direction of the Z axis, which is less than the aperture angle of microscope objective 3, represents the included angle between the projection of the wave vector of the illumination light in the XOY plane and the positive direction of the X axis.

[0034] For ease of expression, it is assumed here that the amplitude of the illumination light is 1. After sample 2 interacts with the oblique plane wave, a certain distribution of the object light field is generated in space, which is represented at the front focal plane of microscope objective 3 as (1) , where represents the oblique illumination light at z = 0.

[0035] The object light field is composed of the illumination field which is not affected by the sample, and the scattering field which carries the sample information, where is between 0 and 1, which is determined by the scattering degree of sample 2. Since sample 2 in the present microscopic system is weakly scattering, can be approximated to 1. Based on this, the object light field represented by formula (1) can also be represented as (2) After being magnified and imaged by the confocal system composed of the microscope objective 3 and the tube lens 4, the object light field shown in formula (2) is propagated to the confocal plane of the tube lens 4 and the first thin lens 6. During this period, the illumination field and the scattered field successively undergo the spatial Fourier transform of the microscope objective 3 and the tube lens 4, and their spectrum is limited by the pupil diameter of the microscope objective 3. Therefore, at the confocal plane of the tube lens 4 and the first thin lens 6, the object light field is expressed as: (3) in, represents the focal length of the microscope objective 3, represents the focal length of the tube lens 4, represents the scattered field with limited spectrum after being amplified and imaged, represents the convolution operation, It represents the complex amplitude point spread function of the confocal system composed of the microscope objective 3 and the tube lens 4. It is essentially the light field distribution generated at the back focal plane of the tube lens 4 after the pupil of the microscope objective 3 is transformed by the spatial Fourier transform of the tube lens 4. Therefore, The spectrum cutoff frequency is At the same time, a field of view limiting module 5 is provided at the confocal plane of the barrel lens 4 and the first thin lens 6, and its light-transmitting area is a side length of b Therefore, the light field distribution shown in formula (3) is further expressed as: (4) in, (5) Subsequently, after being imaged by the confocal system composed of the first thin lens 6 and the second thin lens 8, the light field shown in formula (4) is propagated to the back focal plane of the second thin lens 8. During this period, the spectrum of the light field shown in formula (4) is modulated by the linear polarizer 7. The angle between the polarization direction of the linear polarizer 7 and the positive direction of the X axis ( ) is greater than the angle with the positive direction of the Y axis, such as Figure 2 Therefore, at the back focal plane of the second thin lens 8, the light field includes a distribution-limited X-polarized object light field and a distribution-limited Y-polarized object light field, which can be expressed by the Jones vector as follows: (6) in, represents the angle between the polarization direction of the linear polarizer 7 and the positive direction of the X axis, f 1 represents the focal length of the first thin lens 6, f 2 represents the focal length of the second thin lens 8, (7) At the same time, a first phase-type spatial light modulator 10 is arranged at the back focal plane of the second thin lens 8, and a first phase modulation pattern loaded on the working surface of the first phase-type spatial light modulator 10 is represented as: (8) wherein, NA represents a numerical aperture of the microscopic objective 3, and respectively represent the projections of the unit vector along the wave vector direction of the illumination light on the X-axis and the Y-axis.

[0036] The first phase-type spatial light modulator 10 of the present embodiment only acts on the light field linearly polarized along the Y-axis direction, and does not act on the light field linearly polarized along the X-axis direction. Therefore, after the modulation action of the first phase-type spatial light modulator 10, the light field becomes: (9) Then, after the light splitting action of the first non-polarizing beam splitter 9 and the spatial Fourier transform action of the third thin lens 11, the spectrum of the light field represented by formula (9) is presented at the back focal plane of the third thin lens 11, which is represented as: (10) wherein, represents a two-dimensional spatial Fourier transform; represents the spatial coordinates corresponding to the spectral coordinates; the upper represents the two-dimensional spatial Fourier transform of the corresponding variable, for example, represents the two-dimensional spatial Fourier transform of represents a convolution operation. In addition, and The expressions of and are respectively: (11) It should be noted that, is essentially a two-dimensional rectangular function, which can be further represented as: (12) wherein, represents a rectangular function, which takes the value of 1 in the range of , and takes the value of 0 in other areas. Therefore, the spatial Fourier transform of the two-dimensional rectangular function represented by formula (12) is represented as: (13) Here, represents an oscillation function, which takes 0 at the position of . Therefore, the spectral distribution represented by formula (13) is at and The value at position is 0; and In the area with and of the and In the region of , it is in attenuated oscillation and eventually tends to 0. In practical applications, the spectrum distribution shown in formula (13) can be ignored. and In addition, according to the spatial Fourier transform of the lens, the spatial frequency at the back focal plane of the third thin lens 11 is and spatial coordinates exist and Therefore, the spectrum distribution shown in formula (13) The effective spatial range is and In the quantitative phase microscopy system of the present invention, the effective spatial range of the spectrum distribution shown in formula (13) is approximately and Therefore, the spectrum distribution shown in formula (13) appears as a focused light spot, which is consistent with the actual physical phenomenon.

[0037] For the object light field shown in formula (3), its two-dimensional Fourier transform is expressed as: (14) in, express function, (15) therefore, (16) Based on this, formula (10) can be further expressed as: (17) From formula (17), it can be seen that the object light field linearly polarized along the X-axis direction is not modulated by the first phase-type spatial light modulator 10, and its spectrum (X-polarization spectrum) appears as a circular area (X-polarization circular), and its center coincides with the system optical axis (the back focus of the third thin lens 11). At the same time, the cutoff frequency of the X-polarization spectrum is In addition, the X-polarized illumination field is focused on The object light field linearly polarized along the Y axis is modulated by the first phase-type spatial light modulator 10, and its spectrum (Y polarization spectrum) appears as a circular area deviating from the optical axis of the system (referred to as the Y polarization circle). The Y polarization spectrum and the X polarization spectrum contain exactly the same information, but the former is relative to the latter along the vector A lateral shift occurs. The cut-off frequency of the Y-polarization spectrum is also , but the Y-polarization illumination field is focused at . Therefore, the spectrum (focal point) of the Y-polarization illumination field is located at the edge position of the X-polarization circular, and the spectrum (focal point) of the Y-polarization illumination field, the spectrum (focal point) of the X-polarization illumination field, and the center of the X-polarization circular are located on a straight line, as shown in Figure 3 .

[0038] This shows that, after the modulation by the first phase-type spatial light modulator 10, the lateral wave vector frequency of the Y-polarization illumination field is exactly equal to the cut-off frequency of the X-polarization spectrum. It should be noted that, in the process of formula derivation, the quasi-plane wave generated by the light-emitting diode is approximated as a plane wave, and then the spectrum of the X-polarization illumination field and the spectrum of the Y-polarization illumination field are both ideally considered as infinitely small focal points (δ(x, y) functions). However, the actual light-emitting diode has a certain size (about 5 mm), and therefore the actual spectrum of the X-polarization illumination field and the spectrum of the Y-polarization illumination field are both focal points with a certain size, and in the present application, the diameter of the focal point is about 50 μm. In addition, the limited light transmission area of the field-of-view limiting module 5 causes the spectrum of the X-polarization illumination field and the spectrum of the Y-polarization illumination field (focal points with a diameter of about 50 μm) to be subjected to the convolution of the field-of-view limiting module 5, but the diameters of the spectrum of the X-polarization illumination field and the spectrum of the Y-polarization illumination field in space are not much different from the effective size of the field-of-view limiting module 5, and therefore the spectrum range of the X-polarization illumination field and the spectrum range of the Y-polarization illumination field (the diameter of the focal point) are almost not affected by the convolution of the field-of-view limiting module 5. ) but the diameters of the spectrum of the X-polarization illumination field and the spectrum of the Y-polarization illumination field in space are not much different from the effective size of the field-of-view limiting module 5, and therefore the spectrum range of the X-polarization illumination field and the spectrum range of the Y-polarization illumination field (the diameter of the focal point) are almost not affected by the convolution of the field-of-view limiting module 5.

[0039] Meanwhile, the second phase-type spatial light modulator 13 is arranged at the back focal plane of the third thin lens 11 and is loaded with a pre-designed second phase modulation pattern. The second phase-type spatial light modulator 13 only acts on the light field linearly polarized along the Y-axis direction, and does not act on the light field linearly polarized along the X-axis direction. Therefore, at the back focal plane of the third thin lens 11, only the Y-polarization spectrum is subjected to the modulation action of the second phase-type spatial light modulator 13, and the X-polarization spectrum is only subjected to the mirror reflection action of the second phase-type spatial light modulator 13. In the present embodiment, the second phase modulation pattern loaded by the second phase-type spatial light modulator 13 is divided into two regions: a circular region covering the spectrum (focal point) of the Y-polarization illumination field and other regions. The phase values loaded by the circular region covering the spectrum (focal point) of the Y-polarization illumination field are all 0, and the other regions are loaded with a pre-designed phase modulation pattern. Therefore, the second phase modulation pattern loaded by the second phase-type spatial light modulator 13 is expressed by spatial coordinates as:​​​​​ (18) wherein, f 3 represents the focal length of the third thin lens 11, b represents the side length of the light-transmitting region of the field-of-view limiting module 5, is the radius of the circular region covering the spectrum of the Y-polarized illumination field, The value of d is determined by the actual situation, and in the present application, it is initially set to 25 microns, which can be adjusted according to the actual situation. Therefore, for the spectrum of the Y-polarized illumination field and the X-polarized spectrum, the second phase-type spatial light modulator 13 only plays a role of mirror reflection. In addition, considering the spatial frequency and the spatial coordinate have and a relationship, therefore the spectrum of the Y-polarized illumination field and the X-polarized spectrum are uniformly expressed by the spatial coordinate as: (19) Subsequently, after the light-splitting effect of the second non-polarized light-splitting prism 12 and the spatial Fourier transform effect of the fourth thin lens 14, the spectrum of the light field shown in formula (19) is present at the back focal plane of the fourth thin lens 14, which is expressed by the spatial coordinate as: (20) wherein, ; ; represents the cutoff frequency of the microscope objective 3; represents the Y-polarized illumination field, here ; represents the X-polarized object light field. It can be seen from formula (20) that the distribution of the Y-polarized illumination field and the X-polarized object light field is limited in the same way, and their effective range is a square with a side length of b / M and the geometric center coincides with the optical axis of the system.

[0040] For the spectrum of the Y-polarized scattering field, the second phase-type spatial light modulator 13 performs predetermined phase modulation thereon. Therefore, the spectrum of the Y-polarized scattering field in formula (17) becomes: (21) Then, after the light-splitting effect of the second non-polarized light-splitting prism 12 and the spatial Fourier transform effect of the fourth thin lens 14, the spectrum of the light field shown in formula (21) propagates to the back focal plane of the fourth thin lens 14, which is expressed by the spatial coordinate as: (22) Therefore, it can be seen from equation (22) that the distribution of the Y-polarized scattering field is limited at the back focal plane of the fourth thin lens 14, and its effective range is a square with a side length of b / M , but its whole deviates from the optical axis of the system by a distance of b / M。 Therefore, there is no overlap between the limited Y-polarized scattering field and the limited Y-polarized illumination field and the X-polarized object light field at the back focal plane of the fourth thin lens 14, as shown in Figure 4 Meanwhile, the polarization camera 15 is arranged at the back focal plane of the fourth thin lens 14, and its effective working surface only covers the Y-polarized illumination field and the X-polarized object light field. Therefore, the limited Y-polarized scattering field is not detected by the polarization camera 15. A single exposure of the polarization camera 15 can simultaneously detect linearly polarized light fields in four directions, i.e., 0 degrees (X-axis), 45 degrees, 90 degrees (Y-axis), and 135 degrees. Among them, the light field in the 90-degree direction corresponds to the limited Y-polarized illumination field, i.e., , and its intensity distribution is represented as ; while the linearly polarized light field in the 45-degree direction (which is 45 degrees with the positive direction of the X-axis and the positive direction of the Y-axis at the same time) is represented as: (23) , and its intensity distribution is represented as: (24) It can be seen from equation (23) that at the back focal plane of the fourth thin lens 14, the Y-polarized illumination field and the X-polarized object light field are coherently superimposed in a direction that is 45 degrees with the positive direction of the X-axis and the positive direction of the Y-axis at the same time. Therefore, the actual polarization direction of the Y-polarized illumination field and the X-polarized object light field reaching the back focal plane of the fourth thin lens 14 is 45 degrees with the positive direction of the X-axis and the positive direction of the Y-axis at the same time. The spectral cutoff frequency of the X-polarized object light field is , and the transverse wave vector frequency of the Y-polarized illumination field is also . Therefore, although the transverse wave vector frequency of the illumination field irradiated onto the sample is less than the cutoff frequency of the objective lens, the light field regulation method proposed in the present application makes the transverse wave vector frequency of the Y-polarized illumination field propagating to the back focal plane of the fourth thin lens 14 exactly equal to the spectral cutoff frequency of the X-polarized object light field. It is worth mentioning that since the angle between the polarization direction of the linear polarizer 7 and the positive direction of the X-axis ( ) is greater than the angle with the positive direction of the Y-axis, and for a weak scattering sample, the energy of the illumination field is much greater than the energy of the scattering field, therefore, at the back focal plane of the fourth thin lens 14, the energy of the Y-polarized illumination field can be ensured to be greater than the energy of the X-polarized object light field.

[0041] In general, after being modulated by the first phase-type spatial light modulator 10 and the second phase-type spatial light modulator 13, the Y polarized scattered field deviates from the effective detection range of the polarization camera 15. With X-polarized object light field Coaxial coherent superposition is performed in the direction of 45 degrees to the positive direction of the X axis and the positive direction of the Y axis, and both are within the effective detection range of the polarization camera 15. In addition, the Y polarized illumination field The energy is greater than the X-polarized light field The energy of the Y polarized illumination field The transverse wave vector frequency is exactly equal to the X-polarized object light field The cutoff frequency of the spectrum.

[0042] Then, construct a complex function represented as: (25) in, represents the natural logarithm function, obviously, ,therefore is square integrable. Furthermore, formula (25) can be expanded using Taylor series as follows: (26) Since formula (26) is about A power series of , so The analyticity of The analytical decision of For example, it is further expressed as: (27) Performing a two-dimensional Fourier transform on both sides of formula (27) yields: (28) Apparently, The two-dimensional Fourier transform of is the X-polarized object light field The two-dimensional Fourier transform of Along vector The result after translation. The cutoff frequency is .

[0043] Now establish a temporary rectangular coordinate system , Axis positive direction and The direction is consistent, Axis positive direction and Then, the two-dimensional space coordinates It can be expressed as ,in, and It is along Axis positive direction and The unit vector in the positive direction of the axis. In addition, the space coordinate The corresponding spectrum coordinates are expressed as Obviously, the spectrum shown in formula (28) is distributed in The values ​​in the negative half plane are all 0. According to Titschmarch theory, exist Analytical analysis in the positive half plane. Furthermore, we can know that exist Then, according to the KK relationship, The real part of and the imaginary part The following relationship exists: (29) in, represents the Cauchy principal value. Since the KK relation is a special Hilbert transform, formula (29) can be obtained through the directional Hilbert transform: (30) in, {·} represents the two-dimensional Fourier transform, {·} represents the two-dimensional inverse Fourier transform, represents a symbolic function, which is The value is 1 in the region. The value is 0 in the area, and The value in the region is -1. Therefore, as long as you know The real part of , then its imaginary part It can be obtained by formula (30). Finally, the X-polarized object light field can be calculated as: (31) It is worth noting that The real part of can be derived as Combining formula (24), we can get Therefore, the polarization intensity distribution in the 90-degree and 45-degree directions recorded by the polarization camera 15 can be obtained. The distribution of , and then use formula (30) to obtain For the distribution of For example, its amplitude is equal to , and its transverse wave vector - Can be achieved through The spectrum of the X-polarized object light field is obtained by analyzing the spectrum of the spectrum of the Y-polarized illumination field, which is not described here. Finally, the X-polarized object light field shown in formula (31) is expressed as: (32)

[0044] So far, although the transverse wave vector frequency of the illumination field irradiated by a certain light-emitting diode on the sample is less than the cutoff frequency of the detection objective, the quantitative phase microscopy system of the present application makes the transverse wave vector frequency of the Y-polarized illumination field propagating to the back focal plane of the fourth thin lens 14 exactly equal to the spectral cutoff frequency of the X-polarized object light field through the modulation of the two phase-type spatial light modulators, so that the object light field distribution generated by the sample under the oblique illumination of a certain light-emitting diode is quantitatively obtained through the KK relationship.

[0045] Then, all the light-emitting diodes on the illumination module 1 are sequentially turned on (only one light-emitting diode is turned on at the same time), and the above operation is repeated, so that the X-polarized object light field generated by the sample 2 under the oblique illumination of a plane wave at different angles ( m =1,2,3… N ) is obtained. If two-dimensional fast quantitative phase imaging of the sample is required, only oblique illumination of the sample with four light-emitting diodes is required, which are uniformly distributed on the illumination module 1, i.e. N =4. Then, the X-polarized object light fields ( m =1,2,3,4) obtained are subjected to two-dimensional spatial Fourier transform, spectral shift, spectral superposition, two-dimensional inverse spatial Fourier transform, and phase taking, etc. conventional operations, so that a high-resolution two-dimensional quantitative phase image is obtained, and the specific details are not described here.

[0046] If three-dimensional refractive index imaging of the sample is required, first, the X-polarized object light field generated by the sample under the oblique illumination of a plane wave at different angles ( m =1,2,3… N ) is obtained. Then, the sample is moved laterally so that there is no sample structure in the imaging field of view, and the above operation is repeated to obtain a series of corresponding X-polarized illumination fields ( m =1,2,3… N ). Further, the X-polarized scattering field generated by the sample under the oblique illumination of a plane wave is obtained, i.e., ( m =1,2,3… N ). Finally, the three-dimensional spectral projection of the obtained X-polarized scattering field and the subsequent conventional operations according to the Fourier diffraction tomography theory can obtain the three-dimensional refractive index distribution of the sample, and the specific details are not described here.

[0047] From formula (1) to formula (32), it can be seen that the large numerical aperture quantitative phase microscopy system based on the KK relationship proposed by the present invention uses two phase-type spatial light modulators to respectively perform polarized phase modulation on the object light field and its spectrum, effectively solving the difficult problem that the transverse wave vector frequency of the illumination field must be strictly consistent with the cutoff frequency of the detection objective lens in coaxial quantitative phase microscopy imaging based on the KK relationship. The present invention is applicable to all types of objective lenses. The microscopy system of the present invention does not require complex iterative calculations. It only needs to use Hilbert transform and single exposure to obtain the object light field distribution generated by the sample under oblique illumination at a certain angle. Therefore, the microscopy system of the present invention has very high temporal resolution and imaging robustness. The microscopy system of the present invention has a simple structure, high imaging quality, high spatial resolution, low cost, high spatial bandwidth product, and high stability. Therefore, the present invention can perform label-free, high-resolution two-dimensional rapid quantitative phase imaging and three-dimensional refractive index imaging of fine structures such as subcellular organelles in living cells, and can perform label-free, high-resolution, high-stability and high-speed in situ quantitative detection of samples. It has very good scalability in structure and function, and has great application value in fields such as life science research and industrial testing.

[0048] In order to further demonstrate the feasibility of the large numerical aperture quantitative phase microscopy system based on the KK relationship proposed in the present invention, the specific structure of a set of embodiment 1 is listed here, and the selected device models and parameters are as follows. It should be noted that the large numerical aperture quantitative phase microscopy system based on the KK relationship is not limited to the following example.

[0049] Specifically, the lighting module 1 of this embodiment is composed of 24 light-emitting diodes evenly distributed in a ring. These 24 light-emitting diodes are of identical model, have a wavelength range of 470 nm ± 20 nm, and a diameter of 5 mm. Sample 2 is a weakly scattering sample such as living cells; the microscope objective 3 is an oil immersion objective with a magnification of 100X and a numerical aperture of 1.44 (plan achromatic objective); the focal length of the tube lens 4 is 200 mm and has an apochromatic correction function; the light transmission area of ​​the field of view limiting module 5 is 4 mm. The 4 mm square area is made of an opaque shading plate; the first thin lens 6, the second thin lens 8, the third thin lens 11 and the fourth thin lens 14 are all two-inch double-cemented achromatic lenses with a focal length of 200 mm; the linear polarizer 7 is an economical thin film polarizer, and the wavefront deformation of the light wave after passing through it is less than 1 / 4 of the central wavelength; the first non-polarizing beam splitter prism (9) and the second non-polarizing beam splitter prism 12 are both beam splitting cubes with a beam splitting ratio of 50:50; the first phase-type spatial light modulator 10 and the second phase-type spatial light modulator 13 are of the same model, and their effective pixels are 1920 1152, the size of a single pixel is , the phase modulation resolution is 8 bits; the pixel size of the polarization camera 15 is , its effective pixels are 2464 2056, with a frame rate of 163 frames per second.

[0050] In order to intuitively demonstrate the effectiveness of the present invention, we used the large numerical aperture quantitative phase microscopy system based on the KK relationship to perform two-dimensional quantitative phase imaging of living COS7 cells. Figure 5 and Figure 6 As shown. In order to ensure more uniform spatial resolution, 24 light-emitting diodes were used for multi-angle oblique illumination when performing two-dimensional quantitative phase imaging of living COS7 cells based on the present invention. In the experiment, the effective numerical aperture of the oblique illumination generated by each light-emitting diode was 0.7. Therefore, based on the device of the above example, the lateral spatial resolution of the microscope system of the present invention reaches 220 nanometers, which is sufficient to observe subcellular organelles in living cells. Then, the 24 light-emitting diodes on the lighting module 1 are sequentially lit (only one light-emitting diode is lit at the same time), and the above operation is repeated to obtain a two-dimensional high-resolution quantitative phase image of living COS7 cells. Among them, Figure 5 The spectral distribution of the intensity diagram of the coherent superposition of the Y-polarized illumination field and the X-polarized object light field at a 45-degree angle to both the positive X-axis and the positive Y-axis is shown. The spectral edge of the X-polarized object light field can be clearly seen, and the transverse wave vector frequency of the Y-polarized illumination field can be easily obtained. Then, Figure 6 Quantitative phase images of living COS7 cells are presented, demonstrating the clear and high-resolution restoration of fine structures within these cells, including mitochondria, nuclei, lipid droplets, and pseudopodia. In particular, the distinct morphologies and states of mitochondria are clearly visualized, demonstrating that the microscope system of the present invention possesses sufficiently high spatial resolution and image contrast to enable label-free, high-resolution quantitative phase imaging of subcellular organelles within living cells.

[0051] In summary, the large numerical aperture quantitative phase microscopy system and method based on the KK relationship proposed in the present invention are suitable for different types of objective lenses, especially large numerical aperture oil-immersion objective lenses, etc., and can perform label-free, high-resolution, high-stability and high-speed in-situ quantitative detection of samples. It has very good scalability in structure and function, and has great application value in fields such as life science research and industrial detection.

[0052] In several embodiments provided in the present application, it should be understood that the disclosed devices and methods can be implemented in other ways. For example, the above-described device embodiments are only illustrative, for example, the division of the modules is only a logical function division, and actual implementation can have another division manner, for example, multiple modules or components can be combined or integrated into another system, or some features can be ignored or not executed.

[0053] In addition, each function module in each embodiment of the present application can be integrated into a processing module, or each module can exist physically alone, or two or more modules can be integrated into one module. The integrated module can be realized in the form of hardware or in the form of a hardware plus software function module.

[0054] The above is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application cannot be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application, and all of them should be regarded as falling within the protection scope of the present application.

Claims

1. A large numerical aperture quantitative phase microscopy system based on the KK relationship, characterized in that: The device comprises an illumination module (1), a microscope objective lens (3), a tube lens (4), a field of view limiting module (5), a first thin lens (6), a linear polarizer (7), a second thin lens (8), a first phase-type spatial light modulator (10), a third thin lens (11), a second phase-type spatial light modulator (13), a fourth thin lens (14), and a polarization camera (15), which are arranged in sequence. The sample (2) is arranged at the front focal plane of the microscope objective lens (3), and the illumination module (1) is capable of emitting non-polarized and partially coherent quasi-plane waves for performing transmissive oblique illumination on the sample (2) to generate an object light field, wherein the object light field includes an illumination field that is not affected by the sample and a scattered field that carries sample information; The microscope objective lens (3) and the tube lens (4) have a confocal plane, the field of view limiting module (5) is arranged at the confocal plane of the tube lens (4) and the first thin lens (6), and the field of view limiting module (5) is used to spatially limit the illumination field and the scattering field from the tube lens (4) to obtain an illumination field and a scattering field with limited distribution; The first thin lens (6) and the second thin lens (8) form a confocal system, and the linear polarizer (7) is used to decompose the distribution-limited illumination field and the scattered field into two mutually orthogonal X-polarization components and Y-polarization components, wherein the X-polarization component includes the X-polarized illumination field and the X-polarized scattered field, and the Y-polarization component includes the Y-polarized illumination field and the Y-polarized scattered field; The first phase-type spatial light modulator (10) is arranged at the back focal plane of the second thin lens (8), and is loaded with a pre-designed first phase modulation pattern, and is used to modulate the Y-polarized illumination field and the Y-polarized scattered field; The front focal plane of the third thin lens (11) coincides with the working plane of the first phase-type spatial light modulator (10); the second phase-type spatial light modulator (13) is arranged on the back focal plane of the third thin lens (11) and is loaded with a pre-set second phase modulation pattern for phase modulation of the Y-polarized scattered field; The front focal plane of the fourth thin lens (14) coincides with the working plane of the second phase-type spatial light modulator (13), and the polarization camera (15) is arranged at the back focal plane of the fourth thin lens (14) to obtain the intensity distribution of the illumination field of the sample and the hologram distribution under the illumination of the illumination module (1).

2. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 1, characterized in that: The lighting module (1) comprises a plurality of light-emitting diodes evenly distributed in a ring shape, the plurality of light-emitting diodes being capable of being lit in sequence, and only one light-emitting diode being lit at a time; When a light-emitting diode is turned on, it emits an unpolarized and partially coherent quasi-plane wave.

3. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 2, characterized in that: The field of view limiting module (5) comprises a light-transmitting area and a light-shielding area, wherein: The light-transmitting area is located at the center of the field of view limiting module (5), is in the shape of a square, and the geometric center of the light-transmitting area coincides with the optical axis of the system; The light-shielding area surrounds the periphery of the light-transmitting area and is used to spatially limit the illumination field and the scattering field from the tube lens (4), thereby obtaining an illumination field and a scattering field with limited distribution.

4. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 2, characterized in that: The linear polarizing plate (7) is arranged at a position close to the second thin lens (8), the polarization direction of the linear polarizing plate (7) is perpendicular to the Z axis, and the angle between the polarization direction of the linear polarizing plate (7) and the positive direction of the X axis is greater than the angle between the polarization direction of the linear polarizing plate (7) and the positive direction of the Y axis, wherein the Z axis coincides with the optical axis of the system, and the positive direction of the Z axis is along the light propagation direction; the X axis and the Y axis are perpendicular to each other and perpendicular to the Z axis; The linear polarizer (7) is used to decompose the distribution-limited illumination field and scattered field into an X-polarized illumination field and an X-polarized scattered field and a Y-polarized illumination field and a Y-polarized scattered field.

5. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 2, characterized in that: An OXYZ three-dimensional space coordinate system is established with the object focus of the microscope objective lens (3) as the origin, wherein the Z axis coincides with the system optical axis, the X axis and the Y axis are perpendicular to each other and both perpendicular to the Z axis, and the phase modulation pattern loaded on the working surface of the first phase-type spatial light modulator (10) is expressed as: ， in, m Indicates the serial number of the light emitting diode on the lighting module (1); represents the wave number in vacuum; represents the refractive index of the medium matching the microscope objective (3), represents the central wavelength of the illumination light emitted by the illumination module (1), represents the focal length of the microscope objective (3), represents the focal length of the tube lens (4), represents the focal length of the first thin lens (6), represents the focal length of the second thin lens (8), NA represents the numerical aperture of the microscope objective (3), It represents the angle between the projection of the wave vector of the illumination light in the XOY plane and the positive direction of the X axis. and They represent the projections of the unit vector along the wave vector direction of the illumination light onto the X-axis and Y-axis respectively.

6. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 2, characterized in that: It also includes a first non-polarization beam splitter prism (9) and a second non-polarization beam splitter prism (12), wherein: The first non-polarization beam splitter prism (9) is arranged between the second thin lens (8) and the first phase-type spatial light modulator (10), and is used to transmit the light beam from the second thin lens (8) to the first phase-type spatial light modulator (10), and reflect the light beam from the first phase-type spatial light modulator (10) to the third thin lens (11); The second non-polarizing beam splitter prism (12) is arranged between the third thin lens (11) and the second phase-type spatial light modulator (13), and is used to transmit the light beam from the third thin lens (11) to the second phase-type spatial light modulator (13), and reflect the light beam from the second phase-type spatial light modulator (13) to the fourth thin lens (14).

7. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 5, characterized in that: The second phase modulation pattern loaded by the second phase-type spatial light modulator (13) includes two areas: a circular area covering the spectrum of the Y-polarized illumination field and other areas. The expression of the second phase modulation pattern is: in, represents the focal length of the third thin lens (11), b represents the side length of the light-transmitting area of ​​the field-of-view limiting module (5), is the radius of the circular region covering the spectrum of the Y-polarized illumination field.

8. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 4, characterized in that: The effective working surface of the polarization camera (15) covers the Y polarization illumination field, the X polarization illumination field and the X polarization scattering field, but does not cover the Y polarization scattering field; A single exposure of the polarization camera (15) can simultaneously provide light intensity distribution in four polarization directions: 0 degree, 45 degree, 90 degree, and 135 degree, wherein 0 degree is along the X-axis direction and 90 degree is along the Y-axis direction; The light intensity distribution in the 90-degree direction is the light intensity distribution of the Y-polarized illumination field, and the light intensity distribution in the 45-degree direction is the hologram distribution after the Y-polarized illumination field and the X-polarized object light field are coherently superimposed in a polarization direction that is 45 degrees to both the positive X-axis and the positive Y-axis.

9. The large numerical aperture quantitative phase microscopy system based on the KK relationship according to claim 8, characterized in that: The energy of the Y-polarized illumination field is greater than the energy of the X-polarized object light field, and the transverse wave vector frequency of the Y-polarized illumination field is equal to the spectrum cutoff frequency of the X-polarized object light field.

10. A large numerical aperture quantitative phase microscopy method based on the KK relationship, characterized in that: include: S1: Using the large numerical aperture quantitative phase microscopy system according to any one of claims 2 to 9, obtain the intensity distribution and hologram distribution of the illumination field generated by the sample under the oblique illumination of different light-emitting diodes, and quantitatively obtain the X-polarized object light field distribution generated by the sample under the oblique illumination of different light-emitting diodes using the KK relationship; S2: According to the X-polarized object light field distribution generated by the sample under the oblique illumination of different light-emitting diodes, a two-dimensional quantitative phase image of the sample or a three-dimensional refractive index distribution of the sample is obtained.

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