Three-dimensional imaging system and method based on mixed scattered field and polarization phase contrast detection

Abstract

The invention discloses a three-dimensional imaging system based on mixed scattered field and polarization phase contrast detection, and the system comprises a vertical illumination module, a first thin lens, a non-polarization beam splitting prism, a lens cone lens, a microscope objective, a second thin lens, a linear polarizer, a phase-type spatial light modulator, a third thin lens, and a polarization camera. The vertical illumination module is used for generating non-polarized and partially coherent divergent light; the first thin lens collimates the divergent light and enables a collimated light beam to enter the non-polarization splitting prism at a preset inclination angle, and the collimated light beam is irradiated to the slide and the sample to be measured through the lens cone lens and the microscope objective and generates mirror surface irradiation light and a two-dimensional backscattering field; the second thin lens, the linear polarizer, the phase-type spatial light modulator, the third thin lens and the polarization camera are sequentially arranged on the side, away from the lens barrel lens, of the non-polarization splitting prism. According to the invention, the in-situ three-dimensional refractive index of the sample to be detected can be detected in an unmarked, high-speed, high-quality and high-stability manner.

Description

Technical Field

[0001] The present invention belongs to the technical field of three-dimensional imaging, and in particular relates to a three-dimensional imaging system and method based on mixed scattered field and polarization phase contrast detection. Background Art

[0002] Optical microscopes play an indispensable role in industrial inspection and life science research. Fluorescence microscopy provides researchers with a unique perspective for observing fine structures within living cells through specific fluorescent labels and spatial resolution beyond the diffraction limit. However, the phototoxicity and photobleaching caused by fluorescence excitation are not conducive to the long-term observation of multiple organelles in living cells. Phase contrast microscopy is a label-free imaging technology that converts the phase distribution of transparent samples into intensity modulation. It is widely used in biomedical research and industrial inspection. Its basic principle is to apply specific phase modulation to the non-scattered components of the object light wave, thereby converting undetectable phase information into a measurable intensity distribution. Due to the use of common-path interference optical structure, phase contrast microscopy exhibits significant anti-interference ability. Although this technology can perform high-contrast imaging of transparent samples, it cannot quantitatively obtain the phase information of the sample.

[0003] Quantitative phase microscopy is an emerging label-free imaging technique that visualizes transparent samples by recovering the phase delay caused by the illumination beam passing through the sample. In recent years, researchers have proposed a variety of quantitative phase microscopy techniques, such as digital holography, spatial light interference microscopy, Fourier stacking microscopy, quantitative phase microscopy based on the intensity transfer equation, quantitative differential phase contrast microscopy, and holographic microscopy based on the Kramers–Kronig relationship. It is worth noting that the two-dimensional phase image recovered by these quantitative phase microscopy techniques is the integral of the three-dimensional refractive index distribution of the sample along the axial direction, and therefore lacks three-dimensional imaging capabilities. Since the internal structure of the sample is usually distributed in three dimensions, three-dimensional label-free imaging technology can accurately detect the structure of the sample without affecting its state.

[0004] Optical diffraction tomography (ODT) is a pioneer in 3D label-free imaging, capable of acquiring the three-dimensional refractive index distribution of a sample. It integrates scanning illumination into conventional off-axis digital holographic microscopy to obtain the scattering potential distribution of a sample along different directions, thereby reconstructing the sample's 3D refractive index. ODT has rapidly developed and is widely used in life science research and industrial inspection. However, the mechanical scanning in conventional ODT not only introduces vibration but also affects imaging speed. Furthermore, the off-axis interferometric optical structure significantly reduces system stability. To improve the performance of ODT, researchers have conducted in-depth research from multiple perspectives. First, researchers coupled structured illumination generated by digital micromirror devices (DMDs) or spatial light modulators (SLMs) into conventional ODT to enhance its temporal resolution. Second, researchers enhanced ODT image quality by coupling a low-coherence light source into ODT based on two DMDs or a SLM. Recently, researchers have improved the stability of optical diffraction tomography by combining structured illumination generated by a spatial light modulator with broadband common-path digital holographic microscopy. However, the degree of scattering from the sample significantly affects the imaging quality of this technique. Therefore, optical diffraction tomography based on digital holography still cannot effectively guarantee imaging speed, quality, and stability simultaneously.

[0005] In recent years, researchers have developed a variety of techniques to achieve high-speed, high-quality, and highly stable three-dimensional (3D) label-free imaging. These include 3D imaging techniques based on low-coherence shearing interferometry, 3D imaging techniques based on partially coherent illumination engineering, 3D imaging techniques based on diffraction approximation models and optimization algorithms, white-light diffraction tomography based on spatial light interferometry microscopy, and optical diffraction tomography based on the Kramers–Kronig relationship. These techniques offer high stability, low noise, simple structure, and high cost-effectiveness, enabling high-speed, high-quality, and highly stable 3D label-free imaging of samples. However, these techniques have limitations in their structure and algorithms, and currently cannot achieve high-resolution label-free imaging of subcellular organelles within living cells. For example, optical diffraction tomography based on the Kramers–Kronig relationship requires the effective numerical aperture of the illumination light to be at least as large as that of the detection objective, making it unsuitable for objectives with large numerical apertures. Recently, researchers have proposed a quantitative phase-contrast tomography microscopy technique based on annular mechanical scanning illumination. This technique can obtain the three-dimensional refractive index distribution of a sample without axial scanning, enabling label-free, high-quality, and highly stable three-dimensional tomographic imaging of subcellular organelles within living cells. However, the mechanical scanning and phase shifting operations limit the imaging speed. Furthermore, this method uses phase-contrast modulation to obtain the forward scattered field of the sample, making it unsuitable for strongly scattering samples such as thick tissues.

[0006] In general, there is currently no method that can perform high-speed, high-quality, and highly stable three-dimensional label-free refractive index imaging of subcellular organelles in living cells. In addition, fluorescence imaging plays an indispensable role in three-dimensional label-free refractive index imaging, and the two complement each other. However, there is currently no technology that can achieve sub-pixel matching of fluorescence and three-dimensional refractive index dual-modality imaging. Secondly, the existing three-dimensional label-free refractive index imaging technologies are all based on the forward scattering field. However, the backscattering field is crucial for high axial resolution, high contrast three-dimensional label-free refractive index imaging, but there is currently no method to achieve it. Although a backscattering-based epi-illumination quantitative phase microscopy device has been proposed, it still obtains a two-dimensional phase image and cannot obtain the three-dimensional refractive index distribution of the sample. Summary of the Invention

[0007] To address the above-mentioned problems in the prior art, the present invention provides a three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection. The technical problem to be solved by the present invention is achieved through the following technical solutions: The present invention provides a three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection, comprising an epi-illumination module, a first thin lens, a non-polarizing beam splitter prism, a tube lens, a microscope objective lens, a second thin lens, a linear polarizer, a phase-type spatial light modulator, a third thin lens and a polarization camera, wherein: The epi-illumination module is used to generate non-polarized and partially coherent divergent light in different directions at different times; The first thin lens is used to collimate the divergent light and to make the collimated light beam incident on the non-polarizing beam splitter prism at a preset inclination angle. The tube lens and the microscope objective lens are sequentially arranged in the reflection direction of the non-polarizing beam splitter prism. The non-polarizing beam splitter prism is used to reflect a portion of the collimated light beam to the tube lens. The back focal plane of the barrel lens coincides with the back focal plane of the first thin lens, the front focal plane of the barrel lens coincides with the back focal plane of the microscope objective lens, and the sample to be measured is arranged at the front focal plane of the microscope objective lens through a glass slide, wherein the collimated light beam passes through the barrel lens and the microscope objective lens and is sequentially irradiated onto the glass slide and the sample to be measured, and the glass slide generates specular irradiation light, and the sample to be measured generates a two-dimensional backscattered field; The second thin lens, the linear polarizer, the phase-type spatial light modulator, the third thin lens, and the polarization camera are sequentially arranged on a side of the non-polarizing beam splitter prism away from the tube lens, the front focal plane of the second thin lens coincides with the back focal plane of the tube lens, and the phase-type spatial light modulator is arranged at a common focal plane of the second thin lens and the third thin lens; a phase modulation pattern is provided on the phase-type spatial light modulator, which applies a phase modulation of 0.5π only to the mirror illumination light, and applies a phase modulation of 1.5π to the two-dimensional backscattered field in other areas; The working surface of the polarization camera is located at the back focal plane of the third thin lens.

[0008] Another aspect of the present invention provides a three-dimensional imaging method based on hybrid scattered field and polarization phase contrast detection, comprising: S1: sequentially lighting up each light-emitting diode of the first ring illuminator in the three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection, and obtaining four first interference intensity images when each light-emitting diode is lit using a polarization camera; S2: Obtain the total spectrum distribution of the scattering potential of the sample to be tested based on the four first interference intensity images when each light-emitting diode is lit, and if the sample to be tested is a strong scattering sample, obtain the refractive index distribution of the sample to be tested using the total spectrum distribution of the scattering potential.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes a three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection, which have the following advantages: 1. It is suitable for oil immersion objective lenses and can obtain the three-dimensional refractive index distribution of the sample under test without specific labeling or three-dimensional scanning of the sample under test, thereby performing high-contrast and high-resolution three-dimensional refractive index imaging of the sample under test; 2. It realizes self-interference tomography based on the backscattered field for the first time, and combines it with quantitative phase contrast tomography based on the forward scattered field to achieve three-dimensional refractive index imaging over a wide spectral range, which is applicable to samples under test with different scattering degrees; 3. The speed and image quality of three-dimensional refractive index imaging are greatly improved by the multi-channel polarization detection technology of the polarization camera, the partially coherent scanning illumination of the light-emitting diode, and the phase modulation multiplexing strategy based on the spatial light modulator; 4. The optical structure of common-path interference realizes label-free three-dimensional imaging with excellent stability; 5. It can perform high-speed, high-quality, and highly stable three-dimensional label-free refractive index imaging of subcellular organelles in living cells or biological tissue samples under test; 6. By utilizing the selective spectroscopic principle of a dichroic mirror, it realizes sub-pixel-level matching fluorescence and three-dimensional refractive index dual-modality imaging with a single camera for the first time. Therefore, the three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection of the present invention can perform label-free, high-speed, high-quality, and highly stable in-situ three-dimensional refractive index detection of the sample to be tested, and has very good scalability in structure and function, and has great application value in fields such as industrial detection and life science research.

[0010] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of an optical path of a three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection provided by an embodiment of the present invention; Figure 2 is a schematic diagram of a first multimode optical fiber coupler provided by an embodiment of the present invention; Figure 3 It is the reflection of the irradiated light by the glass slide supporting the sample to be measured when using an oil immersion objective; Figure 4 It is the reflection of the irradiated light by the glass slide supporting the sample to be measured when using a water immersion objective or an air objective; FIG5 is a spectrum distribution of the scattering potential of the sample to be measured on the spherical crown under a certain epi-illumination oblique illumination; Figure 6 It is the total spectrum distribution of the scattering potential of the sample under 30 epi-illumination oblique illuminations (called the total epi-illumination spectrum distribution); Figure 7 It is the phase modulation pattern that the phase-type spatial light modulator (11) should theoretically load when a certain light-emitting diode performs epi-illumination oblique illumination; Figure 8 Based on the phase modulation multiplexing strategy proposed in the present invention, 30 light-emitting diodes share a phase modulation pattern when performing epi-oblique illumination; Figure 9 1 is a schematic structural diagram of another three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection provided by an embodiment of the present invention; Figure 10 is a schematic diagram of the transmitted illumination module; FIG11 is a spectrum distribution of the scattering potential of the sample to be measured on the spherical crown under a certain transmissive oblique illumination; Figure 12 It is the total spectrum distribution of the scattering potential of the weakly scattering sample under 30 transmissive oblique illuminations (called the total transmission spectrum distribution); Figure 13 It is the total spectrum distribution obtained by linearly superimposing the total transmission spectrum distribution and the total incident spectrum distribution of the same sample to be tested; Figure 14 Schematic diagram of the structure of another label-free three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection provided by an embodiment of the present invention; Figure 15 The three-dimensional refractive index distribution of living COS7 cells is restored using the patent of this invention; Figure 16 It is a dual-modality imaging of fluorescence and three-dimensional refractive index with sub-pixel matching in living COS7 cells.

[0012] Description of reference numerals: 1-first ring illuminator; 2-first industrial lens; 3-first multimode fiber coupler; 4-first thin lens; 5-non-polarizing beam splitter; 6-tube lens; 7-microscope objective; 8-sample to be measured; 9-second thin lens; 10-linear polarizer; 11-phase-type spatial light modulator; 12-third thin lens; 13-polarization camera; 14-transmitted illumination module; 14-1-second ring illuminator; 14-2-second industrial lens; 14-3-second multimode fiber coupler; 14-4-fourth thin lens; 14-5-fifth thin lens; 14-6-illumination objective; 15-fluorescence illumination module; 16-sixth thin lens; 17-dichroic mirror; 18-filter. DETAILED DESCRIPTION

[0013] To further illustrate the technical means and effects employed by the present invention to achieve the intended purpose, a three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection according to the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0014] The aforementioned and other technical contents, features, and effects of the present invention are clearly presented in the following detailed description of the specific embodiments in conjunction with the accompanying drawings. Through the description of the specific embodiments, a deeper and more specific understanding of the technical means and effects adopted by the present invention to achieve the intended purpose can be obtained. However, the accompanying drawings are provided for reference and illustration purposes only and are not intended to limit the technical solutions of the present invention.

[0015] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations are intended to cover non-exclusive inclusion, such that an article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of additional identical elements in the article or device comprising the element.

[0016] Example 1 See Figure 1 , Figure 1This is a schematic diagram of the optical path of a three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection provided by an embodiment of the present invention. The three-dimensional imaging system includes an epi-illumination module, a first thin lens 4, a non-polarizing beam splitter prism 5, a tube lens 6, a microscope objective 7, a second thin lens 9, a linear polarizer 10, a phase-type spatial light modulator 11, a third thin lens 12 and a polarization camera 13, wherein the epi-illumination module is used to generate non-polarized and partially coherent divergent light in different directions at different times; the first thin lens 4 is used to collimate the divergent light and make the collimated light beam incident on the non-polarizing beam splitter prism 5 at a preset inclination angle; the tube lens 6 and the microscope objective 7 are sequentially arranged in the reflection direction of the non-polarizing beam splitter prism 5; the non-polarizing beam splitter prism 5 is used to reflect a portion of the collimated light beam to the tube lens 6; the back focal plane of the tube lens 6 coincides with the back focal plane of the first thin lens 4, the front focal plane of the tube lens 6 coincides with the back focal plane of the microscope objective 7, and the sample to be measured 8 is set on the microscope objective through a glass slide. At the front focal plane of the non-polarizing beam splitter 5, the collimated light beam passes through the tube lens 6 and the microscope objective lens 7 and is sequentially irradiated onto the glass slide and the sample to be measured 8, and the glass slide generates mirror illumination light, and the sample to be measured 8 generates a two-dimensional backscattering field; the second thin lens 9, the linear polarizer 10, the phase-type spatial light modulator 11, the third thin lens 12 and the polarization camera 13 are sequentially arranged on the side of the non-polarizing beam splitter 5 away from the tube lens 6, the front focal plane of the second thin lens 9 coincides with the back focal plane of the tube lens 6, and the phase-type spatial light modulator 11 is arranged at the confocal plane of the second thin lens 9 and the third thin lens 12; a phase modulation pattern is provided on the phase-type spatial light modulator 11, which only applies a phase modulation of 0.5π to the mirror illumination light, and applies a phase modulation of 1.5π to the two-dimensional backscattering field in other areas; the working surface of the polarization camera 13 is located at the back focal plane of the third thin lens 12.

[0017] Furthermore, the epi-illumination module of the present embodiment includes a first annular illuminator 1, a first industrial lens 2 and a first multimode fiber coupler 3, wherein the first annular illuminator 1 includes a plurality of light-emitting diodes arranged in a ring, the plurality of light-emitting diodes are exactly the same, and can be lit in sequence to generate non-polarized and partially coherent inclined divergent light; the first industrial lens 2 is used to collect and scale the inclined divergent light and then couple it to the first multimode fiber coupler 3; the first multimode fiber coupler 3 includes a plurality of identical multimode optical fibers, the input ports and output ports of the plurality of multimode optical fibers are respectively evenly distributed on two circular rings to form an input ring and an output ring, and the axes of the input ring and the output ring coincide with the axis of the first industrial lens 2; the number of multimode optical fibers is the same as the number of light-emitting diodes, so that the first m The light emitted by the first LED is m Multimode optical fibers are used for coupling.

[0018] Preferably, see Figure 2 , Figure 2Schematic diagram of a first multimode fiber coupler provided by an embodiment of the present invention. The first ring illuminator 1 of this embodiment is composed of 30 identical light-emitting diodes, which are used to perform epi-type oblique illumination on the sample 8 to be tested. The 30 light-emitting diodes are illuminated in sequence, and at any time only one light-emitting diode is in the illuminated state, and all other light-emitting diodes remain in the off state. For ease of description, Figure 1 Only the light path propagation when a certain light emitting diode is lit is drawn. Specifically, when a certain light emitting diode is lit, it emits non-polarized and partially coherent small-angle divergent light. This divergent light can be approximately regarded as a collimated light beam, which is collected and scaled by the first industrial lens 2 and then coupled into a multimode optical fiber in the first multimode fiber coupler 3. The first industrial lens 2 of this embodiment is a small focal length industrial lens. It should be noted that Figure 2 The figure only schematically illustrates one structure of the first multimode fiber coupler 3 and does not limit its other structural forms. The output ring of the first multimode fiber coupler 3 is located at the front focal plane of the first thin lens 4. Therefore, the divergent light emitted from the output port of a certain multimode optical fiber is collimated by the first thin lens 4 and then incident on the non-polarizing beam splitter prism 5 in the optical path at a certain inclination angle. After the beam splitting effect of the non-polarizing beam splitter prism 5, 50% of the collimated light is reflected. The reflected collimated light beam is collected by the tube lens 6 and converged to the confocal plane of the tube lens 6 and the microscope objective lens 7. The light beam converged at the back focal plane of the microscope objective lens 7 forms a quasi-plane wave after the collimation effect of the microscope objective lens 7. The quasi-plane wave is irradiated onto the sample to be measured 8 located at the front focal plane of the microscope objective lens 7 at a certain inclination angle. Part of the illumination light directly passes through the glass slide supporting the sample 8 to interact with the sample 8 to generate a light field with a certain distribution in space, while the other part of the illumination light is reflected by the mirror surface of the bottom surface of the glass slide and then collected by the microscope objective lens 7. Figure 1 、 Figure 3 and Figure 4 As shown. Among them, Figure 3 Indicates the reflection of the illumination light by the glass slide when using an oil immersion objective. At this time, only the upper surface of the glass slide will undergo specular reflection, and a quasi-plane wave (i.e., specular illumination light) will be generated that propagates in a mirror-symmetrical manner. Figure 4 The light emitting diodes used in the device of the present invention generate partially coherent light, and their coherence length is less than 10 microns, which is much shorter than the thickness of the glass slide. Therefore, only the coherence length is used here. Figure 3 It should be noted that when the microscope objective lens 7 is an oil-immersion objective lens or a water-immersion objective lens, the angle between the wave vector of the irradiating light in the glass slide and the optical axis of the system must be smaller than the critical angle of the glass slide relative to the medium surrounding the sample to be measured, otherwise total internal reflection will occur and the sample to be measured 8 will not be effectively illuminated.

[0019] In order to ensure the spatial resolution of the imaging system, the angle between the wave vector of the illuminating light in the glass slide and the optical axis of the system should be as large as possible if conditions permit. The light field generated by the interaction between the sample to be tested 8 and the illuminating light that directly passes through the glass slide includes three parts: the forward illuminating light (i.e., quasi-plane wave) that is not affected by the sample to be tested 8, the forward scattered field along the direction of the forward illuminating light wave vector, and the backscattered field in the direction opposite to the forward illuminating light wave vector, wherein the forward illuminating light that is not affected by the sample to be tested and the forward scattered field along the direction of the forward illuminating light wave vector are both far away from the system and are not detected by the system, while the backscattered field in the direction opposite to the forward illuminating light wave vector is collected by the microscope objective 7. Therefore, under the epi-oblique illumination, only the backscattered field and the quasi-plane wave (mirror illuminating light) that propagates symmetrically with the mirror surface are collected by the microscope objective 7 and subsequently propagated, such as Figure 1 and Figure 3 As shown. Specifically, the two-dimensional backscattered field and the mirror illumination light generated by the sample 8 to be tested and the glass slide at the front focal plane of the microscope objective 7 are propagated to its rear focal plane under the action of the spatial Fourier transform of the microscope objective 7. At this time, the spectrum information of the two-dimensional backscattered field is distributed at the rear focal plane of the microscope objective 7 and is limited by its limited aperture (pupil diameter), while the mirror illumination light is converged at the rear focal plane of the microscope objective 7. The mirror illumination light appears as a converging spot of a certain size at the rear focal plane of the microscope objective 7. The converging spot has a certain lateral offset relative to the optical axis of the system, and the distance of the lateral offset is related to the wave vector of the mirror illumination light and the focal length of the microscope objective 7.

[0020] Furthermore, the spectral distribution of the two-dimensional backscattered field limited by the pupil aperture and the converged mirror illumination light are magnified and imaged by the confocal system composed of the tube lens 6 and the second thin lens 9, and then reach the confocal plane of the second thin lens 9 and the third thin lens 12. During this period, they are modulated by the linear polarizer 10. Moreover, due to the splitting effect of the non-polarizing beam splitter prism 5, only 50% of the light energy is received by the second thin lens 9. The linear polarizer 10 is placed near the second thin lens 9, and the polarization direction of the linear polarizer 10 is 45 degrees to the positive direction of the X-axis and the positive direction of the Y-axis at the same time, and the positive direction of the X-axis and the positive direction of the Y-axis are perpendicular to each other and parallel to the sample surface of the sample to be measured (8). Preferably, as Figure 1 The positive direction of the X-axis is vertically upward in the paper, and the positive direction of the Y-axis is perpendicular to the paper and outward.

[0021] Therefore, at the confocal plane of the second thin lens 9 and the third thin lens 12, the spectral distribution of the two-dimensional backscattered field (limited by the pupil aperture) and the converged mirror illumination light (with a convergent spot of a certain size) are both linearly polarized (the polarization direction is 45 degrees to both the positive direction of the X-axis and the positive direction of the Y-axis), and they can both be divided into a linear polarization component along the X-axis direction and a linear polarization component along the Y-axis direction with equal power. At the same time, a phase-type spatial light modulator 11 is provided at the confocal plane of the second thin lens 9 and the third thin lens 12. In the present invention, it only acts on linear polarized light along the X-axis direction. A phase modulation pattern is provided on the phase-type spatial light modulator 11, which only applies a 0.5 to the converged mirror illumination light (the linear polarization component along the X-axis direction). Phase modulation of 1.5 is applied to other regions (linear polarization components along the X-axis direction). Phase modulation. After the selective phase modulation of the phase-type spatial light modulator 11 and the spatial Fourier transform of the third thin lens 12, the spectrum-limited two-dimensional backscattered field and the mirror-illuminated light (quasi-plane wave) are superimposed on the back focal plane of the third thin lens 12 and recorded by the polarization camera 13 set at this position. Each pixel of the polarization camera 13 is composed of four sub-pixels, which are used to detect linear polarized light in the directions of 0 degrees, 45 degrees, 90 degrees and 135 degrees respectively. Therefore, one exposure of the polarization camera 13 can provide four interference intensity maps at the same time. After a simple calculation, the two-dimensional backscattered field generated by the sample to be tested 8 at the front focal plane of the microscope objective 7 can be obtained under this epi-illumination oblique illumination. According to the Fourier diffraction tomography theory, the spectral distribution of the obtained two-dimensional backscattered field is projected, and the spectral distribution of the scattering potential of the sample to be tested 8 can be obtained on the corresponding spherical crown, as shown Figure 5 shown.

[0022] Subsequently, the 30 light-emitting diodes on the first ring illuminator 1 are switched in sequence, and the above operation is repeated, and finally the total spectrum distribution of the scattering potential of the sample 8 to be tested under 30 epi-illumination oblique illuminations (called epi-illumination total spectrum distribution) can be obtained, as shown in FIG. Figure 6 In order to show the spectrum distribution more intuitively, Figure 6 The displayed spectrum is the result of sectioning the original spectrum (remove K x Positive space and K y The intersection space of the positive space). Then, through spatial inverse Fourier transform and simple mathematical operations, the refractive index distribution of the sample under test with high axial resolution can be obtained. Therefore, the 3D imaging system shown in this embodiment can perform 3D label-free refractive index imaging not only on thin, weakly scattering samples under test, but also on strongly scattering samples that cannot be detected by traditional transmission quantitative phase contrast tomography microscopy.

[0023] It is worth noting that the closer the mirror illumination light is to a plane wave, the smaller the diameter of the spot that converges on the working surface of the phase-type spatial light modulator 11 will be. Then the quality of the obtained two-dimensional backscattered field will also be improved, thereby improving the accuracy of the refractive index reconstruction of the sample to be measured. To this end, this embodiment uses a multimode optical fiber to couple each light emitting diode to make the mirror illumination light as close to a plane wave as possible. Theoretically, under the epi-illumination of a certain light emitting diode, the phase-type spatial light modulator 11 only applies 0.5 Phase modulation is applied to other spectral regions, while 1.5 Phase modulation. In this way, the phase-type spatial light modulator 11 needs to load 30 phase patterns. However, the phase-type spatial light modulator 11 needs a certain amount of time to switch the liquid crystal during the pattern loading process, which usually takes about 20 milliseconds. Therefore, it takes at least 0.6 seconds to load 30 phase modulation patterns, which is not conducive to high-speed three-dimensional label-free refractive index imaging. In order to ensure imaging speed, the present invention proposes a phase modulation multiplexing strategy based on the spatial light modulator, that is, 30 light-emitting diodes share a phase modulation pattern when performing epi-oblique illumination. Please refer to Figure 7 and Figure 8 ,in, Figure 7 It is the phase modulation pattern that the phase-type spatial light modulator 11 should theoretically load when a certain light-emitting diode performs epi-illumination oblique illumination; Figure 8 This is based on the phase modulation multiplexing strategy proposed in this embodiment of the present invention. The 30 LEDs share a phase modulation pattern when performing epi-oblique illumination. Experimental results will be compared and verified later, so we will not elaborate on them here. Thus, in this embodiment, the phase-type spatial light modulator 11 only needs to load one phase modulation pattern, which takes only 20 milliseconds.

[0024] From the optical path propagation of this embodiment, it can be seen that the backscattered field generated by the sample to be tested 8 and the mirror illumination light generated by the glass slide pass through exactly the same optical device from beginning to end. Therefore, the three-dimensional imaging system of this embodiment has very high stability. The three-dimensional imaging system of this embodiment greatly improves the speed and image quality of three-dimensional refractive index imaging through the multi-channel polarization detection technology of the polarization camera, the partially coherent scanning illumination of the light-emitting diode, and the phase modulation multiplexing strategy based on the spatial light modulator. In addition, the epi-illumination oblique illumination performed by 30 light-emitting diodes enables the refractive index distribution of the obtained sample to be tested to have high lateral spatial resolution and axial spatial resolution. Therefore, the three-dimensional imaging system of this embodiment can achieve high-speed, high-quality, high-stability, high-spatial resolution and high-contrast self-interference tomography (three-dimensional label-free refractive index imaging) for samples to be tested with different scattering degrees through spectral projection of the backscattered field without the need for specific labeling or three-dimensional scanning of the sample to be tested.

[0025] Example 2 Based on the first embodiment, this embodiment also provides another three-dimensional imaging system based on mixed scattered field and polarization phase contrast detection, such as Figure 9 As shown. This embodiment adds a transmission illumination module (14) on the basis of the first embodiment, aiming to implement rapid quantitative phase contrast tomography based on the forward scattered field, thereby realizing high-speed, high-quality, and highly stable label-free three-dimensional refractive index imaging based on mixed scattered field and polarization phase contrast detection. Therefore, the three-dimensional imaging system of this embodiment can successively perform quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the back scattered field on the same sample to be tested, and finally realize three-dimensional refractive index imaging based on a large spectrum range. In this embodiment, the quantitative phase contrast tomography part based on the forward scattered field and the self-interference tomography part based on the back scattered field share all the devices of the detection light path, including the microscope objective lens 7, the tube lens 6, the second thin lens 9, the linear polarizer 10, the phase-type spatial light modulator 11, the third thin lens 12 and the polarization camera 13.

[0026] like Figure 10 As shown, the transmitted lighting module 14 of this embodiment includes a second ring illuminator 14-1, a second industrial lens 14-2, a second multimode fiber coupler 14-3, a fourth thin lens 14-4, a fifth thin lens 14-5 and an illumination objective lens 14-6, which are arranged in sequence. The second ring illuminator 14-1 includes a plurality of light-emitting diodes arranged in a ring, and the plurality of light-emitting diodes are exactly the same and can be lit in sequence to generate non-polarized and partially coherent inclined divergent light; the second industrial lens 14-2 is used to illuminate the second ring illuminator. The inclined divergent light generated by 14-1 is collected and scaled and then coupled to the second multimode fiber coupler 14-3; the second multimode fiber coupler 14-3 includes a plurality of identical multimode optical fibers, the input ports and output ports of the plurality of multimode optical fibers are evenly distributed on two rings to form an input ring and an output ring, and the axes of the input ring and the output ring coincide with the axis of the second industrial lens 14-2; the number of multimode optical fibers in the second multimode fiber coupler 14-3 is the same as the number of light-emitting diodes in the second ring illuminator 14-1, so that the second multimode fiber coupler 14-3 has the same number of multimode optical fibers as the light-emitting diodes in the second ring illuminator 14-1. m The light emitted by the first LED is m Multimode optical fibers are used for coupling.

[0027] First, rapid quantitative phase contrast tomography based on the forward scattered field is performed on the sample to be tested 8. Specifically, the second ring illuminator 14-1 of the present embodiment is composed of 30 identical light-emitting diodes, which are intended to perform transmissive oblique illumination on the sample to be tested. These 30 light-emitting diodes are exactly the same as the 30 light-emitting diodes on the first ring illuminator 1. They are lit in sequence, and only one light-emitting diode is in the lit state at any time, and all other light-emitting diodes remain off. When a certain light-emitting diode on the second ring illuminator 14-1 is lit, it emits non-polarized and partially coherent small-angle divergent light, which is collected and scaled by the second industrial lens 14-2 and coupled into a multimode optical fiber in the second multimode fiber coupler 14-3. The structure of the second multimode fiber coupler 14-3 is the same as that of the first multimode fiber coupler 3, and it is composed of 30 identical multimode optical fibers. The input ports and output ports of these 30 multimode optical fibers are evenly distributed on two circular rings to form an input ring and an output ring, respectively, as shown in FIG. Figure 2 It should be noted that although the structure of the second multimode fiber coupler 14-3 is the same as that of the first multimode fiber coupler 3, the diameters of their input ring and output ring are not necessarily the same. The second industrial lens 14-2 of this embodiment is a small focal length industrial lens.

[0028] The divergent light emitted from the output port of a multimode optical fiber propagates through the confocal system composed of the fourth thin lens 14-4 and the fifth thin lens 14-5, and then converges to the confocal plane of the fifth thin lens 14-5 and the illumination objective lens 14-6, and converges within the pupil diameter range of the illumination objective lens 14-6. After the converged light beam is collimated by the illumination objective lens 14-6, it is obliquely irradiated on the sample to be tested 8 located at the confocal plane of the illumination objective lens 14-6 and the microscope objective lens 7 in the form of a quasi-plane wave. At this time, the sample to be tested is a weakly scattering sample to be tested, such as Figure 9 and 10 It should be noted that the effective numerical aperture of the obliquely illuminated quasi-plane wave is smaller than the numerical aperture of the microscope objective 7. In order to ensure the spatial resolution of the imaging device, the effective numerical aperture of the obliquely illuminated quasi-plane wave should be as large as possible when conditions permit.

[0029] The light field generated by the interaction between the weakly scattered sample to be tested 8 and the obliquely illuminated quasi-plane wave includes three parts: ① forward illumination light (i.e., quasi-plane wave) that is not affected by the sample to be tested 8, ② forward scattered field along the wave vector direction of the forward illumination light, and ③ backscattered field in the direction opposite to the wave vector direction of the forward illumination light, wherein the backscattered field in the direction opposite to the wave vector direction of the forward illumination light is away from the microscope objective 7 and is not detected by the system, while the forward illumination light that is not affected by the sample to be tested and the forward scattered field along the wave vector direction of the forward illumination light are collected by the microscope objective 7. Specifically, the forward scattered field and the forward illumination light generated at the front focal plane of the microscope objective 7 propagate to its back focal plane under the action of the spatial Fourier transform of the microscope objective 7. At this time, the spectral information of the two-dimensional forward scattered field is distributed at the back focal plane of the microscope objective 7 and is limited by its limited aperture, while the forward illumination light converges at the back focal plane of the microscope objective 7. The forward illuminating light appears as a converging spot of a certain size at the rear focal plane of the microscope objective 7. The converging spot has a certain lateral offset relative to the system optical axis, and the lateral offset distance is related to the wave vector of the forward illuminating light and the focal length of the microscope objective 7.

[0030] Furthermore, the spectral distribution of the two-dimensional forward scattered field (limited by the pupil aperture) and the converged forward illumination light are magnified and imaged by the confocal system composed of the tube lens 6 and the second thin lens 9, and then arrive at the confocal plane of the second thin lens 9 and the third thin lens 12. During this period, they are modulated by the linear polarizer 10. Moreover, due to the splitting effect of the non-polarizing beam splitter prism 5, only 50% of the light energy is received by the second thin lens 9. As described in Example 1, the polarization direction of the linear polarizer 10 is 45 degrees to the positive direction of the X-axis and the positive direction of the Y-axis at the same time. Therefore, at the confocal plane of the second thin lens 9 and the third thin lens 12, the spectral distribution of the two-dimensional forward scattered field and the converged forward illumination light are both linearly polarized. Moreover, as described in Example 1, a phase modulation pattern is provided on the phase-type spatial light modulator 11, which only applies 0.5 to the converged forward illumination light. Phase modulation is applied to other regions, while 1.5 Phase modulation. After the selective phase modulation of the phase-type spatial light modulator 11 and the spatial Fourier transform of the third thin lens 12, the spectrum-limited forward scattered field and the forward irradiated light (quasi-plane wave) are superimposed on the back focal plane of the third thin lens 12 and recorded by the polarization camera 13 arranged there. Similar to the first embodiment, one exposure of the polarization camera 13 can provide four interference intensity maps at the same time. After a simple calculation, the two-dimensional forward scattering field generated by the sample to be tested at the front focal plane of the microscope objective 7 can be obtained under the transmitted oblique illumination. According to the Fourier diffraction tomography theory, the spectral distribution of the obtained two-dimensional forward scattering field is projected, and the spectral distribution of the scattering potential of the weakly scattering sample to be tested 8 can be obtained on the corresponding spherical crown, as shown in FIG. Figure 11Then, the 30 light-emitting diodes on the second ring illuminator 14-1 are switched in sequence, and the operation in the above-mentioned second embodiment is repeated, and finally the total spectrum distribution (called the total transmission spectrum distribution) of the scattering potential of the weak scattering sample 8 under 30 transmission-type oblique illuminations can be obtained, as shown in FIG. Figure 12 In order to show the spectrum distribution more intuitively, Figure 12 The displayed spectrum is the result of sectioning the original spectrum (remove Kx Positive space and Ky intersection space of positive spaces).

[0031] Next, the self-interference tomography based on the backscattered field is performed on the same sample to be tested. Specifically, the light emitting diodes on the second ring illuminator 14-1 are turned off, and according to the imaging process described in Example 1, the total spectrum distribution of the scattering potential of the same weakly scattering sample to be tested 8 is obtained under the epi-illumination oblique illumination of the 30 light emitting diodes of the first ring illuminator 1 (referred to as the epi-illumination total spectrum distribution). Then, the total spectrum distribution of the transmission total spectrum distribution and the epi-illumination total spectrum distribution are linearly superimposed to obtain the total spectrum distribution of the scattering potential of the weakly scattering sample to be tested 8, as shown in FIG. Figure 13 In order to show the spectrum distribution more intuitively, Figure 13 The displayed spectrum is the result of sectioning the original spectrum (remove Kx Positive space and Ky The intersection space of the positive space). Then, through spatial inverse Fourier transform and simple mathematical operations, a refractive index distribution of the sample under test with a wide spectral range can be obtained based on the mixed scattered field. Clearly, the 3D imaging system of this embodiment can obtain a spectral distribution over a wider range, thus achieving a refractive index distribution of the sample under test with higher axial spatial resolution and image contrast.

[0032] It is worth noting that during quantitative phase contrast tomography based on the forward scattered field of the sample 8, the closer the forward illumination light approaches a plane wave, the higher the quality of the obtained two-dimensional forward scattered field, and thus, the higher the quality of the obtained refractive index distribution of the sample 8. To this end, in the transmitted illumination module 14, a multimode optical fiber couples each light-emitting diode to ensure that the forward illumination light approaches a plane wave as closely as possible. Furthermore, to ensure imaging speed, the three-dimensional imaging system of this embodiment still utilizes a phase modulation multiplexing strategy based on a spatial light modulator during quantitative phase contrast tomography based on the forward scattered field of the sample 8, with 30 light-emitting diodes sharing a single phase modulation pattern. Thus, when the three-dimensional imaging system of this embodiment performs quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the backscattered field on the same sample, only two phase modulation patterns need to be loaded onto the phase-type spatial light modulator 11, which takes a total of 40 milliseconds.

[0033] Because the 30 LEDs on the second ring illuminator 14-1 are identical to the 30 LEDs on the first ring illuminator 1, the phase-type spatial light modulator 11 responds identically to the light emitted by these 60 LEDs. Based on this, in this embodiment, quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the backscattered field can share a single phase modulation pattern. This phase modulation pattern can be preloaded before data acquisition, so that the time required to load this phase modulation pattern is not included in the total system data acquisition time. Therefore, the imaging speed of the three-dimensional imaging system is determined solely by the exposure time of the polarization camera 13. It should be noted that the various component parameters of the transillumination module 14 need to be adjusted so that the forward illumination light (generated by the second ring illuminator 14-1) and the converged specular illumination light (generated by the first ring illuminator 1) converge at the back focal plane of the microscope objective 7 correspond one-to-one and overlap.

[0034] The three-dimensional imaging system of this embodiment enables quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the back scattered field to share all devices of the detection light path, so that the two modes are seamlessly connected, and for the first time, high-speed, high-quality, and highly stable three-dimensional refractive index imaging based on a large spectrum range is realized. From the light path propagation of this embodiment, it can be seen that under transmissive oblique illumination, the forward scattered field and forward illumination light generated by the weakly scattered sample to be tested 8 pass through exactly the same optical devices at the same time from beginning to end. Under epi-infrared oblique illumination, the back scattered field and mirror illumination light generated by the sample to be tested 8 pass through exactly the same optical devices at the same time from beginning to end. Therefore, the three-dimensional imaging system of this embodiment has very good immunity to external interference, that is, the system of this embodiment has very high stability. Furthermore, the system of this embodiment significantly improves the speed and image quality of three-dimensional refractive index imaging by utilizing multi-path polarization detection technology of a polarization camera, partially coherent scanning illumination of light-emitting diodes, and a phase modulation multiplexing strategy based on a spatial light modulator during the sequential forward-scattered field quantitative phase-contrast tomography and backscattered field self-interference tomography of the same sample. Furthermore, the epi- and trans-illumination oblique illumination provided by the light-emitting diodes enables the refractive index distribution of the sample to be obtained to have high lateral and axial spatial resolution. Therefore, the three-dimensional imaging system of this embodiment can achieve high-speed, high-quality, high-stability, high-spatial-resolution, and high-contrast three-dimensional refractive index imaging of weakly scattering samples such as subcellular organelles within living cells without requiring specific labeling or three-dimensional scanning of the sample.

[0035] Example 3 Based on the second embodiment, this embodiment provides another three-dimensional imaging system based on mixed scattered field and polarization phase contrast detection, such as Figure 14As shown. This embodiment adds a fluorescence illumination module 15, a sixth thin lens 16, a dichroic mirror 17, and a filter 18 to the second embodiment, aiming to achieve sub-pixel matching of fluorescence and three-dimensional refractive index dual-modal imaging. Therefore, the three-dimensional imaging system of this embodiment first performs quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the back scattered field on the sample to be tested 8, achieving three-dimensional refractive index imaging based on a large spectrum range; then, fluorescence imaging is performed on the same sample to be tested 8, ultimately achieving sub-pixel matching of fluorescence and three-dimensional refractive index dual-modal imaging. Of course, the sample to be tested 8 must have the characteristic of being fluorescence-excitable (self-fluorescent or fluorescently labeled). In the system structure of this embodiment, the quantitative phase contrast tomography part based on the forward scattered field, the self-interference tomography part based on the back scattered field, and the fluorescence imaging part share all the components of the detection optical path, including the microscope objective 7, the tube lens 6, the second thin lens 9, the linear polarizer 10, the phase-type spatial light modulator 11, the third thin lens 12, and the polarization camera 13.

[0036] First, based on Example 1 or Example 2, a three-dimensional refractive index distribution of the sample to be tested 8 is obtained. If the sample to be tested 8 is a weakly scattering sample to be tested, the three-dimensional refractive index distribution of the sample to be tested is obtained using the inventive device shown in Example 2; and if the sample to be tested 8 is a strongly scattering sample to be tested, the three-dimensional refractive index distribution of the sample to be tested is obtained using the inventive device shown in Example 1. It should be noted that the dichroic mirror 17 and the optical filter 18 of this embodiment have no effect on the epi-oblique illumination generated by the first annular illuminator 1 and the trans-oblique illumination generated by the second annular illuminator 14-1, that is, the epi-oblique illumination and the trans-oblique illumination directly pass through the dichroic mirror 17 and the optical filter 18 without being affected by them.

[0037] Then, fluorescence imaging is performed on the same sample to be tested. Specifically, the fluorescence illumination module 15 generates the required fluorescence excitation light. Its structure is not limited here. It can be a wide-field fluorescence excitation light path, or a super-resolution fluorescence excitation light path, etc. The exit surface of the fluorescence illumination module 15 is located at the front focal plane of the sixth thin lens 16. Therefore, the fluorescence excitation light generated by the fluorescence illumination module 15 is transmitted through the confocal system composed of the sixth thin lens 16, the dichroic mirror 17 and the microscope objective 7, and reaches the front focal plane of the microscope objective 7, and performs specific fluorescence excitation on the sample to be tested 8 there. During this period, the fluorescence excitation light is reflected by the dichroic mirror 17 and enters the pupil aperture of the microscope objective 7. The fluorescence radiation signal generated by the sample to be tested 8 is imaged by the magnification system composed of the microscope objective 7 and the tube lens 6, and reaches the confocal plane of the tube lens 6 and the second thin lens 9. During this period, the spectrum of the fluorescence radiation signal is limited by the pupil aperture of the microscope objective 7, and the residual fluorescence excitation light is blocked by the filter 18. Filter 18 is located between dichroic mirror 17 and tube lens 6. It only blocks the fluorescence excitation light and does not affect the fluorescence radiation signal generated by the sample 8. Furthermore, dichroic mirror 17 does not affect the fluorescence radiation signal generated by the sample 8. The fluorescence radiation signal then propagates to the front focal plane of the second thin lens 9, through the confocal system composed of the second thin lens 9 and the third thin lens 12, and reaches the back focal plane of the third thin lens 12, where it is recorded by the polarization camera 13.

[0038] During this period, the fluorescence radiation signal undergoes the splitting effect of the non-polarization beam splitter prism 5, and only 50% of the light energy enters the subsequent imaging system. Although the fluorescence radiation signal passes through the linear polarizer 10 and the phase-type spatial light modulator 11, during the fluorescence imaging process, the phase-type spatial light modulator 11 is loaded with a full area of ​​0 The phase modulation pattern is not modulated, and the fluorescence radiation signal is not modulated. Therefore, the four fluorescence radiation intensity maps obtained by the single exposure of the polarization camera 13 are simply linearly superimposed to obtain the fluorescence radiation intensity map produced by the sample under the fluorescence excitation light. Then, appropriate image processing and analysis are performed based on the structure and function of the fluorescence illumination module 15 to obtain the final fluorescence radiation intensity image. Finally, without the need for any image processing, the obtained three-dimensional refractive index distribution map of the sample under test and the fluorescence radiation intensity map are directly coupled to achieve sub-pixel matching fluorescence and three-dimensional refractive index dual-modal imaging.

[0039] Since the quantitative phase contrast tomography part based on the forward scattered field, the self-interference tomography part based on the back scattered field, and the fluorescence imaging part share all devices in the detection optical path, the three-dimensional imaging method provided in this embodiment realizes sub-pixel-level matched fluorescence and three-dimensional refractive index dual-modal imaging using a single camera for the first time.

[0040] Specifically, this embodiment will provide a detailed description of the imaging principles of each imaging component. Regarding the fluorescence imaging component, its imaging principles will not be elaborated here. Simply perform appropriate image processing and analysis based on the structure and function of the fluorescence illumination module 15 to obtain the final fluorescence radiation intensity image. Regarding the quantitative phase contrast tomography component based on the forward scattered field and the self-interference tomography component based on the backscattered field, the imaging principles will be described in detail here.

[0041] For convenience, the front focus of the microscope objective lens 7 is set as the origin O, the vertical upward direction in the paper is the positive direction of the X axis, the vertical outward direction in the paper is the positive direction of the Y axis, and the horizontal right direction in the paper is the positive direction of the Z axis. Figure 1 、 Figure 9 and Figure 14 As shown. A non-uniform sample 8 is placed at the front focal plane of the microscope objective 7, and the sample 8 is n 0 medium. At this time, the refractive index distribution of the sample 8 to be measured is expressed in the set coordinate system OXYZ as ,in, Represents the three-dimensional space coordinates. According to Fourier diffraction tomography theory, under the irradiation of a certain inclined plane wave (the central wavelength of the irradiating light wave is , whose wave number in vacuum is ), the sample 8 to be tested will generate a specific light field distribution in space. Among them, the two-dimensional scattered field generated on the plane z = 0 (the front focal plane of the microscope objective lens) and the scattering potential distribution of the sample 8 to be tested are closely related, and the relationship between them is described by the following formula: (1) in, j represents the imaginary unit, represents the scattering potential distribution The three-dimensional Fourier transform of Represents the scattered field The two-dimensional Fourier transform of It represents the three-dimensional spectrum coordinates of the scattered field generated by the sample under the illumination of the inclined plane wave; is the refractive index of the matching medium of the microscope objective 7; represents a unit vector.

[0042] Since the microscope objective lens in the actual microscope system has a limited numerical aperture, it can only collect the scattered field within a certain frequency range. and Satisfy the inequality ( NA is the numerical aperture of the microscope objective lens 7); in addition, Represents the three-dimensional spectrum coordinates of the scattering potential of the sample 8 to be measured, where represents the wave vector of the inclined plane wave incident on the sample to be measured, and Represents a unit vector, which is determined by the position of the light-emitting diode, etc. As can be seen from formula (1), under the illumination of a certain inclined plane wave, the two-dimensional scattering field generated by the sample to be tested on the plane z = 0 (the front focal plane of the microscope objective 7) has a one-to-one corresponding projection relationship with the three-dimensional spectrum distribution of the scattering potential of the sample to be tested 8 on a specific spherical cap. The position of the spherical cap is determined by the numerical aperture of the microscope objective 7, the refractive index of the matching medium of the microscope objective 7, the central wavelength of the inclined plane wave, and the wave vector of the inclined plane wave. Therefore, by changing the illumination angle of the inclined plane wave and obtaining the two-dimensional scattering field generated by the sample to be tested 8 on the plane z = 0 (the front focal plane of the microscope objective 7) under the corresponding illumination, the three-dimensional spectrum distribution of the scattering potential of the sample to be tested 8 can be obtained on different spherical caps. Finally, the three-dimensional spectrum distribution of the scattering potential on different spherical caps is linearly superimposed, and then a spatial inverse Fourier transform is performed to obtain the scattering potential distribution of the sample to be tested 8, and then the three-dimensional refractive index distribution of the sample to be tested 8 can be obtained.

[0043] Clearly, accurately calculating the two-dimensional scattered field generated by the sample 8 at z = 0 is crucial for achieving three-dimensional, label-free refractive index imaging. Based on this, the present invention proposes a three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection, encompassing quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the backscattered field.

[0044] As for the self-interference tomography based on the backscattered field, as described in the first embodiment, when a certain LED on the first ring illuminator 1 is lit, the inclined quasi-plane wave illuminates the sample 8 located at the front focal plane of the microscope objective 7 at a certain inclination angle. Since the light emitted by the 30 LEDs on the first ring illuminator 1 is partially coherent light, its coherence length is calculated as Lcoh=2 ln(2) λ2 / π / △λ = 2.5 microns (λ = 0.53 microns, representing the central wavelength of the light emitted by the LED, and △λ = 0.05 microns, representing the spectral width of the light emitted by the LED), which is much smaller than the thickness of the glass slide. Therefore, under epi-oblique illumination, the two-dimensional backscattered field generated at the front focal plane of the microscope objective 7 and the quasi-plane wave (specular illumination light) generated by the reflection from the upper surface of the glass slide are coherent, while the quasi-plane wave generated by the reflection from the lower surface of the glass slide is incoherent with the former two. Therefore, under a certain epi-oblique illumination, the light field distribution generated by the sample 8 at the front focal plane of the microscope objective 7 and that can be collected by it is expressed as: (2) in, represents the distribution of quasi-plane waves generated by surface reflection on the glass slide (specular illumination light), and represents the distribution of quasi-plane waves generated by reflection from the lower surface of the glass slide (incoherent mirror illumination light), represents the phase change caused by the thickness of the glass slide, A and B represent the amplitudes of the quasi-plane waves reflected from the upper and lower surfaces of the glass slide, respectively. represents the wave vector of the incident quasi-plane wave irradiating the sample 8 to be measured, and Represents a unit vector. It should be noted that, since the present invention uses a multimode optical fiber to couple the light emitted by each light-emitting diode one-to-one, so that the quasi-plane wave irradiated on the sample 8 to be tested is as close to a plane wave as possible, the quasi-plane wave generated by the reflection from the upper and lower surfaces of the glass slide can be approximately represented as a plane wave. represents the two-dimensional backscattered field generated at the front focal plane of the microscope objective 7. It should be noted that in formula (2) The other two items are irrelevant and are written together for the sake of simplicity. The intensity of the other two items is superimposed, which will be described in detail later. In addition, when the microscope objective lens 7 is an oil-immersion objective lens, only the upper surface of the glass slide has mirror reflection. At this time, the formula (2) Take 0; When the microscope objective lens 7 is a water immersion objective lens or an air objective lens, there is mirror reflection on both the upper and lower surfaces of the glass slide. At this time, the formula (2) Take 1.

[0045] Subsequently, after the spatial Fourier transform of the microscope objective 7, the spectrum information of the light field distribution shown in formula (2) is distributed at the back focal plane of the microscope objective 7 and is limited by its limited aperture. At this time, the light field distribution at the back focal plane of the microscope objective 7 is expressed as: (3) in, represents the two-dimensional backscattered field The spatial Fourier transform of represents the focal length of the microscope objective lens 7, is the pupil function of microscope objective 7, which has a radius of The value is 1 in the circular area and 0 in other areas; Indicated on the back focal plane of microscope objective 7 The unit pulse function at is essentially the converging specular illumination light and the converging incoherent specular illumination light. Since the wave vectors of the specular illumination light and the incoherent specular illumination light have a certain inclination angle relative to the system optical axis (Z axis), the light spots they converge at the back focal plane of the microscope objective 7 have a certain lateral offset relative to the system optical axis.

[0046] The light field distribution shown in formula (3) is magnified and imaged by the confocal system composed of the tube lens 6 and the second thin lens 9, and then reaches the confocal plane of the second thin lens 9 and the third thin lens 12. During this period, the light field is modulated by the linear polarizer 10. Therefore, the light field distribution at the confocal plane of the second thin lens 9 and the third thin lens 12 is expressed by the Jones matrix as follows: (4) in, represents the focal length of the tube lens 6, represents the focal length of the second thin lens 9. Meanwhile, the phase-type spatial light modulator 11 only acts on the linearly polarized light along the X-axis direction, and only applies a 0.5 Phase modulation is applied to other regions, while 1.5 Therefore, after being modulated by the phase-type spatial light modulator 11, the light field distribution shown in formula (4) becomes: (5) Subsequently, after the spatial Fourier transform of the third thin lens 12, the spectrum-limited two-dimensional backscattered field, the specular illumination light, and the incoherent specular illumination light propagate to the back focal plane of the third thin lens 12 and are recorded by the polarization camera 13 set there. At this time, the light field distribution at the back focal plane of the third thin lens 12 is expressed as: (6) in, Represents the convolution operation; represents the focal length of the third thin lens 12; ; represents the complex amplitude point spread function of the three-dimensional imaging system, that is, a unit pulse function at the front focus of the microscope objective 7 The light field distribution generated at the back focal plane of the third thin lens 12 after passing through the pairwise confocal system composed of the microscope objective lens 7, the tube lens 6, the second thin lens 9 and the third thin lens 12. Essentially the pupil function of the microscope objective 7 The light field distribution generated at the back focal plane of the third thin lens 12 after passing through the pairwise confocal system composed of the tube lens 6, the second thin lens 9 and the third thin lens 12. Therefore, The two-dimensional Fourier transform of is a circular function with a radius of The value of is 1 in the circular spectrum area and 0 in other areas. The non-polarizing beam splitter prism 5, the linear polarizer 10, and the phase-type spatial light modulator 11 are not considered in the generation and analysis process. Since each pixel of the polarization camera 13 is composed of four sub-pixels, they are used to detect linear polarized light in the directions of 0 degrees (X-axis direction), 45 degrees, 90 degrees (Y-axis direction), and 135 degrees respectively. Therefore, a single exposure of the polarization camera 13 can provide four interference intensity maps at the same time. In addition, as mentioned above, under epi-type oblique illumination, the two-dimensional backscattered field generated at the front focal plane of the microscope objective 7 and the quasi-plane wave (mirror illumination light) generated by the reflection on the upper surface of the glass slide are coherent, while the quasi-plane wave generated by the reflection on the lower surface of the glass slide is incoherent with the former two. Therefore, the four interference intensity maps obtained after the polarization camera 13 performs a single exposure recording of the light field distribution shown in formula (6) are respectively expressed as: (7) in, represents the distribution of the spectrally limited two-dimensional backscattered field on the working surface of the polarization camera 13, Formula (7) can be further expanded as: (8) in, express The amplitude distribution of ,here, express Phase distribution. Formula (8) is further expanded into: (9) From formula (9), it can be easily obtained: (10) in, It should be noted that when the microscope objective lens 7 is a water immersion objective lens or an air objective lens, Take 1. At this time, it is impossible to accurately obtain Amplitude distribution of , and thus spectrum projection cannot be performed according to Fourier diffraction tomography theory. When the microscope objective lens 7 is an oil immersion objective lens, Take 0, then set: (11) (12) Combining formulas (11) and (12), we can obtain: (13) Then, the amplitude of the specular illumination light generated by the reflection on the upper surface of the glass slide under the epi-illumination can be obtained: (14) Therefore, on the working surface of the polarization camera 13, the spectrum-limited two-dimensional backscattered field distribution can be calculated as: (15) It is worth noting that the amplitude of the two-dimensional backscattered field is proportional to the amplitude of the epi-illumination, while the amplitude of the light emitted by the 30 light-emitting diodes on the first ring illuminator 1 has certain differences. In order to prevent the uneven illumination intensity from causing errors in the refractive index reconstruction of the sample to be measured, formula (15) is corrected to: (16) in, T and R Respectively represent the transmittance and reflectance of the glass slide surface to light, which can be obtained experimentally or from the manufacturer. In this way, the two-dimensional backscattered field distribution generated by the sample to be tested 8 under unit amplitude epi-oblique illumination can be obtained. Then, based on the Fourier diffraction tomography theory described in formula (1), the spectrum distribution of the two-dimensional backscattered field shown in formula (16) is projected, and the three-dimensional spectrum distribution of the scattering potential of the sample to be tested on the corresponding spherical cap under the epi-oblique illumination can be obtained. The 30 light-emitting diodes on the first ring illuminator 1 are sequentially illuminated, and the same sample to be tested is subjected to epi-oblique illumination at different angles. The polarization camera 13 is used to obtain four polarization intensity images under each illumination (formula (9)), and the data processing process of formulas (10) to (16) is repeated to obtain the two-dimensional backscattered field distribution under each illumination. By performing spectrum projection based on formula (1), the total spectrum distribution of the scattering potential of the sample to be tested (episodic total spectrum distribution) can be obtained under 30 epi-oblique illuminations.

[0047] (17) in, m =1, ... ,30 represents the serial numbers of the 30 light-emitting diodes on the first ring illuminator 1. Indicates in m Spectral distribution of the scattering potential of the sample 8 to be measured obtained under the epi-oblique illumination of a light emitting diode.

[0048] For the quantitative phase contrast tomography part based on the forward scattered field, as described in Example 2, it is known that it shares all the devices of the detection light path with the self-interference tomography part based on the backscattered field. When a certain light emitting diode on the second annular illuminator 14-1 is lit, the inclined quasi-plane wave carries out transmission-type oblique illumination to the sample to be tested 8 at the front focal plane of the microscope objective 7 at a certain inclination angle. After the interaction between the sample to be tested 8 and the obliquely illuminated quasi-plane wave, the forward irradiation light (i.e., quasi-plane wave) that is not affected by the sample to be tested is generated, the forward irradiation field along the wave vector direction of the forward irradiation light, and the backscattered field facing the wave vector direction of the forward irradiation light. Among them, only the forward irradiation light that is not affected by the sample to be tested and the forward irradiation field along the wave vector direction of the forward irradiation light are collected by the microscope objective 7. Therefore, under a certain transmission-type oblique illumination, the light field distribution that is generated by the sample to be tested 8 at the front focal plane of the microscope objective 7 and can be collected by it is expressed as follows: (18) in, represents the forward irradiation light (quasi-plane wave) that is not affected by the sample 8 to be tested, represents the amplitude of the forward irradiation light, represents the wave vector of the transmitted quasi-plane wave irradiated on the sample 8 to be measured, and represents a unit vector.

[0049] Since the present invention uses multimode optical fiber to perform one-to-one coupling on the light emitted by each light-emitting diode, so that the quasi-plane wave irradiated on the sample to be tested 8 is as close to a plane wave as possible, the forward irradiated light not affected by the sample to be tested 8 can be approximately expressed as a plane wave. It represents the two-dimensional forward scattered field generated at the front focal plane of the microscope objective 7.

[0050] Then, after the spatial Fourier transform of the microscope objective 7, the spectrum information of the light field distribution shown in formula (18) is distributed at the back focal plane of the microscope objective 7 and is limited by its limited aperture. At this time, the light field distribution at the back focal plane of the microscope objective 7 is expressed as: (19) in, represents the two-dimensional forward scattered field The spatial Fourier transform of . Indicated on the back focal plane of microscope objective 7 The unit pulse function at is essentially the convergent forward illumination light. Since the wave vector of the forward illumination light has a certain inclination angle relative to the system optical axis (Z axis), the convergent forward illumination light has a certain lateral offset relative to the system optical axis at the rear focal plane of the microscope objective 7.

[0051] The light field distribution shown in formula (19) is magnified and imaged by the confocal system composed of the tube lens 6 and the second thin lens 9, and then reaches the confocal plane of the second thin lens 9 and the third thin lens 12. During this period, the light field is modulated by the linear polarizer 10. Therefore, the light field distribution at the confocal plane of the second thin lens 9 and the third thin lens 12 is expressed by the Jones matrix as follows: (20) At the same time, the phase modulation pattern set on the phase-type spatial light modulator 11 only applies 0.5 Phase modulation is applied to other regions, while 1.5 Therefore, after modulation by the phase-type spatial light modulator 11, the light field distribution shown in formula (20) becomes: (twenty one) Then, after the spatial Fourier transform of the third thin lens 12, the spectrum-limited two-dimensional forward scattered field and the forward illumination light propagate to the back focal plane of the third thin lens 12 and are recorded by the polarization camera 13 set there. At this time, the light field distribution at the back focal plane of the third thin lens 12 is expressed as: (twenty two) The four intensity maps obtained by the polarization camera 13 after single exposure recording of the light field distribution shown in formula (22) are respectively expressed as: (twenty three) in, represents the distribution of the spectrum-limited two-dimensional forward scattered field on the working surface of the polarization camera 13; Formula (23) can be further expanded as: (twenty four) in, express The amplitude distribution of ,here, express Phase distribution. Formula (24) is further expanded into: (25) From formula (25), it can be easily obtained: (26) Furthermore, let: (27) (28) Combining formulas (27) and (28), we can obtain: (29) The amplitude of the forward irradiation light under the transmissive oblique illumination can be obtained: (30) Therefore, on the working surface of the polarization camera 13, the spectrum-limited two-dimensional forward scattered field distribution can be calculated as: (31) Since the amplitude of the two-dimensional forward scattered field is proportional to the amplitude of the transmissive oblique illumination, and the amplitudes of the light emitted by the 30 light-emitting diodes on the second ring illuminator 14-1 have certain differences, in order to prevent the uneven illumination intensity from causing errors in the refractive index reconstruction of the sample to be measured, formula (31) is corrected to: (32) In this way, the two-dimensional forward scattering field distribution generated by the sample to be tested 8 under unit amplitude transmissive oblique illumination can be obtained. Then, based on the Fourier diffraction tomography theory described in formula (1), the spectrum distribution of the two-dimensional forward scattering field shown in formula (32) is projected, and the three-dimensional spectrum distribution of the scattering potential of the sample to be tested on the corresponding spherical cap under this transmissive oblique illumination can be obtained. The 30 light-emitting diodes on the second ring illuminator 14-1 are sequentially illuminated, and the same sample to be tested 8 is subjected to transmissive oblique illumination at different angles. The polarization camera 13 is used to obtain four polarization intensity images under each illumination (formula (25)), and the data processing process from formula (26) to formula (32) is repeated to obtain the two-dimensional forward scattering field distribution under each illumination. By implementing spectrum projection with formula (1), the total spectrum distribution of the scattering potential of the sample to be tested (transmissive total spectrum distribution) can be obtained under 30 transmissive oblique illuminations.

[0052] (33) in, n =1, ... ,30 represent the serial numbers of the 30 light emitting diodes on the second ring illuminator 14 - 1 . Indicates in n The spectral distribution of the scattering potential of the sample to be measured is obtained under the transmissive oblique illumination of a light emitting diode.

[0053] If the sample 8 to be tested is a weakly scattering sample such as a living cell, the sample 8 is subjected to self-interference tomography based on the backscattering field and quantitative phase contrast tomography based on the forward scattering field (i.e., the imaging method described in Example 2). The total spectrum distribution of the incident light shown in formula (17) and the total spectrum distribution of the transmission shown in formula (33) are linearly superimposed to obtain the total spectrum distribution of the scattering potential of the sample to be tested. If the sample 8 to be tested is a strong scattering sample such as a thick tissue, only self-interference tomography based on the backscattering field is performed on the sample 8 (i.e., the imaging method described in Example 1). In this case, the total spectrum distribution of the incident light shown in formula (17) is the total spectrum distribution of the scattering potential of the sample to be tested. Finally, the refractive index distribution of the sample to be tested can be obtained by spatial inverse Fourier transform and simple mathematical operations. Therefore, the three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection proposed in the present invention can provide a wider range of spectrum distribution for weakly scattering samples such as living cells, and the obtained refractive index distribution has higher axial spatial resolution and image contrast. At the same time, the present invention can also perform three-dimensional, label-free refractive index imaging of strongly scattering samples, such as thick tissues, that are difficult to detect using conventional transmission-based quantitative phase-contrast tomography microscopy. It should be noted that the self-interference tomography based on the backscattered field of the present invention is only applicable to high-numerical-aperture oil-immersion objective lenses. Therefore, the three-dimensional imaging system of the present invention is only applicable to oil-immersion objective lenses. That is, the microscope objective lens 7 of the present invention is an oil-immersion objective lens.

[0054] As described in Example 1, the 30 light-emitting diodes on the first annular illuminator 1 are evenly distributed about the system optical axis (Z axis) for the 30 angles of epi-illumination implemented on the sample to be measured. Therefore, the angles between the wave vectors of the 30 epi-illuminations and the system optical axis (Z axis) in the matching medium of the immersion oil objective lens are the same, that is, In addition, as described in Example 2, the 30 light-emitting diodes on the second annular illuminator 14-1 provide 30 angles of transmissive oblique illumination to the sample to be measured, which are evenly distributed about the system optical axis (Z axis). Therefore, the angles between the wave vectors of the 30 transmissive oblique illuminations and the system optical axis (Z axis) in the matching medium of the immersion oil objective lens are the same, that is, Therefore, when performing the three-dimensional label-free refractive index imaging described in Example 2 on weakly scattering samples such as living cells, the lateral spatial resolution of the imaging system is calculated as , min{·} means taking the minimum; the axial resolution is calculated as When performing the three-dimensional label-free refractive index imaging described in Example 1 on a sample with strong scattering, such as thick tissue, the lateral spatial resolution of the system is calculated as ; The axial resolution is calculated as Therefore, the three-dimensional imaging system of the present invention has very good spatial resolution, especially very good axial spatial resolution, and can perform high-speed, high-quality, and highly stable three-dimensional label-free refractive index imaging of organelles in living cells.

[0055] Example 4 Based on the above embodiment, this embodiment further provides a three-dimensional imaging method based on mixed scattered field and polarization phase contrast detection, comprising the following steps: S1: sequentially lighting up each light-emitting diode of the first ring illuminator 1 in the three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection described in Example 1, and obtaining four first interference intensity images when each light-emitting diode is lit using the polarization camera 13; S2: Obtain the total spectrum distribution of the scattering potential of the sample to be tested based on the four first interference intensity images when each light-emitting diode is lit, and if the sample to be tested is a strong scattering sample, obtain the refractive index distribution of the sample to be tested using the total spectrum distribution of the scattering potential.

[0056] The method of this embodiment further includes: If the sample to be tested is a weakly scattering sample to be tested, each light-emitting diode of the second ring illuminator 14-1 is lit in sequence, and four second interference intensity maps when each light-emitting diode is lit are obtained using the polarization camera 13; the total transmission spectrum distribution of the scattering potential of the sample to be tested is obtained based on the four second interference intensity maps when each light-emitting diode is lit; the total incident spectrum distribution and the total transmission spectrum distribution are linearly superimposed to obtain the total spectrum distribution of the scattering potential of the sample to be tested, and the refractive index distribution of the sample to be tested is obtained using the total spectrum distribution.

[0057] Specifically, the calculation process of the total incident spectrum distribution, the total transmitted spectrum distribution and the refractive index distribution of the sample to be measured can be found in Example 3 and will not be repeated here.

[0058] It should be pointed out that when performing spectrum projection operation on the obtained two-dimensional backscattered field and two-dimensional forward scattered field, the wave vector corresponding to the epi-illumination and transmissive oblique illumination must be accurately mastered. Before S1, it also includes: determining the wave vector generated by each light-emitting diode in the falling-type illumination module on the sample surface, and determining the phase modulation pattern loaded on the phase-type spatial light modulator based on the phase modulation multiplexing strategy, so that all light-emitting diodes in the falling-type illumination module share a phase modulation pattern; or determining the wave vector generated by each light-emitting diode in the falling-type illumination module on the sample surface and the wave vector generated by each light-emitting diode in the transmission illumination module on the sample surface, so that the transverse wave vector generated by each light-emitting diode in the falling-type illumination module on the sample surface and the transverse wave vector generated by each light-emitting diode in the transmission illumination module on the sample surface are one-to-one and the same, and determining the phase modulation pattern loaded on the phase-type spatial light modulator based on the phase modulation multiplexing strategy, so that all light-emitting diodes in the falling-type illumination module and all light-emitting diodes in the transmission illumination module share a phase modulation pattern. Specifically, the following steps are taken: Step 1: Before performing three-dimensional imaging of the sample 8 based on mixed scattered field and polarization phase contrast detection, a temporary strong scattering sample is placed at the front focal plane of the microscope objective 7, and a temporary lens is placed between the phase-type spatial light modulator 11 and the third thin lens 12 to enable the polarization camera 13 to simultaneously clearly image the pupil plane of the microscope objective 7 and the working plane of the phase-type spatial light modulator 11.

[0059] Step 2: Turn on all the light emitting diodes on the second ring illuminator 14 - 1 and load a phase modulation pattern of all zeros on the phase-type spatial light modulator 11 .

[0060] Step 3: Use the polarization camera 13 to record the current intensity information, and then average the four intensity images recorded by the polarization camera 13 to obtain the position information of the pupil diameter of the microscope objective lens 7, including the center coordinates ( x 0, y 0) and diameter d .

[0061] Step 4: Place the sample 8 to be tested on the front focal plane of the microscope objective 7, and light the first ring illuminator 1. m ( m =1,…,30) light emitting diodes, and a polarization camera 13 is used to record the intensity information under the illumination.

[0062] Step 5: Average the four intensity images recorded in step 4 to obtain the intensity image I bm , the position of the converging specular light can be obtained under this illumination (x m , y m ). Then, mThe transverse wave vector of an epi-illumination oblique illumination is calculated as , and then we can get the m The wave vector of the epi-illumination is expressed as .

[0063] Step 6: Repeat steps 4 to 5 until all the LEDs on the first ring illuminator 1 are lit in sequence. In this way, the wave vectors corresponding to the 30 epi-illumination oblique illuminations can be accurately determined.

[0064] Step 7: Light up the second ring illuminator 14-1. n ( n =1,…,30) light emitting diodes, and a polarization camera 13 is used to record the intensity information under the illumination.

[0065] Step 8: Average the four intensity images recorded in step 7 to obtain the intensity image I tn , the position of the convergent forward illumination light can be obtained under this illumination ( x n , y n ). Then, n The transverse wave vector of a transmissive oblique illumination is calculated as , and then we can get the n The wave vector of the transmitted oblique illumination is expressed as .

[0066] Step 9: Repeat steps 7 to 8 until all the LEDs on the second ring illuminator 14-1 are lit in sequence. In this way, the wave vectors corresponding to the 30 transmissive oblique illuminations can be accurately determined.

[0067] Step 10: Adjust the parameters of each device of the transillumination module 14 and execute step 9 until and At this point, the quantitative phase contrast tomography based on the forward scattered field and the self-interference tomography based on the back scattered field can share a phase modulation pattern (such as Figure 8 shown).

[0068] Step 11: Take the 30 intensity images obtained in step 5 I bm ( m =1,…,30) and perform linear superposition to obtain the intensity image I b , which shows the position information of 30 converging mirror-illuminated lights (30 converging light spots).

[0069] Step 12: Temporarily place a light emitting diode between the tube lens 6 and the second thin lens 9 to uniformly illuminate the working surface of the phase-type spatial light modulator 11 .

[0070] Step 13: Load the phase modulation pattern based on the phase modulation multiplexing strategy on the phase type spatial light modulator 11 ( Figure 8 ), and four polarization intensity images are recorded by the polarization camera 13. The four polarization intensity images are linearly superimposed to obtain the intensity image ISLM, which shows the 30 circular gray areas (0.5 ) location.

[0071] Step 14: Compare ISLM and I b , if the 30 circular gray areas on the image ISLM cannot be perfectly covered one to one I b The positions of the 30 circular gray areas on the phase-type spatial light modulator 11 are adjusted, and the phase modulation pattern based on the phase modulation multiplexing strategy is updated ( Figure 8 ).

[0072] Step 15: Repeat steps 13 to 14 until the 30 circular gray areas on the image ISLM are perfectly covered one by one. I b At this time, the phase-type spatial light modulator 11 performs accurate phase modulation (0.5 ), and quantitative phase contrast tomography based on the forward scattered field and self-interference tomography based on the back scattered field can share a phase modulation pattern.

[0073] Step 16: Remove the temporarily placed light-emitting diode and temporary lens, and conduct a three-dimensional label-free imaging experiment based on hybrid scattered field and polarization phase contrast detection on the sample 8 to be tested.

[0074] It should be noted that if the sample 8 to be tested is strongly scattering, only self-interference tomography based on the backscattered field is performed (i.e., the imaging method described in Example 1). In this case, steps 7 to 10 are not applicable, and only steps 1 to 6 and steps 11 to 16 are performed. If the sample 8 to be tested is weakly scattering, self-interference tomography based on the backscattered field and quantitative phase contrast tomography based on the forward scattered field are performed on the sample to be tested (i.e., the imaging method described in Example 2). In this case, steps 1 to 16 are performed.

[0075] In order to further reflect the feasibility of three-dimensional imaging of the present invention, a set of device models and parameters are listed here as follows. It should be noted that the models and parameters of the devices in the system are not limited to the following description. The 30 light-emitting diodes on the first ring illuminator 1 are exactly the same as the 30 light-emitting diodes of the second ring illuminator 14-1, and the wavelength range is 530±25 nanometers; the main bodies of the first ring illuminator 1 and the second ring illuminator 14-1 are obtained by 3D printing technology; the focal lengths of the first industrial lens 2 and the second industrial lens 14-2 are both 12 mm, and the distance between the former and the first ring illuminator 1 and the distance between the latter and the second ring illuminator 14-1 are both 110 mm; the 30 multimode optical fibers in the first multimode fiber coupler 3 are exactly the same as the 30 multimode optical fibers in the second multimode fiber coupler 14-3, and they are step-index multimode optical fibers. The jumper has a core diameter of 50 microns; the first thin lens 4 is an achromatic doublet with a focal length of 200 mm; the non-polarizing beam splitter 5 is a beam splitter cube with a beam splitting ratio of 50:50; the focal length of the tube lens 6 is 200 mm; the microscope objective 7 is an oil immersion objective with a magnification of 100X and a numerical aperture of 1.44; the second thin lens 9 is an achromatic doublet with a focal length of 250 mm; the wavefront deformation of the light wave after passing through the linear polarizer 10 is less than 1 / 4 wavelength; the phase modulation resolution of the phase-type spatial light modulator 11 is 8 bits; the third thin lens 12 is an achromatic doublet with a focal length of 300 mm; the pixel size of the polarization camera 13 is , its effective pixel is 2464×2056, and the frame rate is 163 fps; the fourth thin lens 14-4 and the fifth thin lens 14-5 are both achromatic doublet lenses with a focal length of 50 mm; the illumination objective lens 14-6 is an air objective lens with a magnification of 100X, a numerical aperture of 0.9, and a working distance of 1 mm; the light source in the fluorescence illumination module 15 consists of two lasers, whose central wavelengths are 488 nm and 561 nm respectively; the sixth thin lens 16 is a tube lens with a focal length of 200 mm; the dichroic mirror 17 is selected as a 488 / 561 nm bilateral laser dichroic beam splitter; the filter 18 is selected as a 488 / 568 nm Yokogawa emission filter. Furthermore, in order to intuitively demonstrate the effectiveness of the device of the present invention, the three-dimensional imaging of living COS7 cells based on mixed scattered field and polarization phase contrast detection was performed using the above-mentioned device. Figure 15As shown. Since the living COS7 cells are weakly scattering samples to be tested, the imaging system described in Example 2 is used to carry out self-interference tomography based on the backscattered field and quantitative phase contrast tomography based on the forward scattered field. Among them, the effective numerical apertures of the 30 light-emitting diodes on the first ring illuminator 1 and the 30 light-emitting diodes on the second ring illuminator 14-1 for the epi-illumination and transmissive oblique illumination of the living COS7 cells located at the front focal plane of the microscope objective 7 are both 0.7. Therefore, the lateral spatial resolution of the above-mentioned system is 247 nanometers, and the axial resolution is 185 nanometers. In addition, in the process of three-dimensional label-free imaging of living COS7 cells, the exposure time of the polarization camera 13 is 3 milliseconds. Therefore, the imaging speed of the above-mentioned system is 1000 / (60*3)=5.5 frames per second. According to the imaging method proposed in the present invention, Figure 15 (a) shows the 3D refractive index distribution of a living COS7 cell with a thickness of 5.2 μm. To demonstrate the 3D imaging capability of the present invention, Figure 15 (b) presents Figure 15 The refractive index distribution of a certain XZ plane (Y=13 μm) in (a), and Figure 15 (c) shows Figure 15 (a) The refractive index distribution of a certain YZ plane (X=26 microns). It can be seen that the three-dimensional refractive index distribution of lipid droplets at different levels is clearly displayed. In addition, Figure 15 (d)-(f) presents Figure 15 The refractive index distribution of the three XY planes (Z = 1.6 μm, Z = 2.2 μm, and Z = 3.2 μm) in (a), and Figure 15 (g)-(i) are Figure 15 The refractive index distribution of the white dotted area in (d)-(f) shows that different lipid droplets focus on different XY planes, indicating that the system and method of the present invention have excellent three-dimensional imaging capabilities. Figure 15 The results in the literature show that the refractive index distribution of living COS7 cells restored by the system and method proposed in the present invention has very good spatial resolution and image contrast. Therefore, the present invention can perform high-speed, high-quality, and highly stable three-dimensional label-free refractive index imaging of organelles in living cells.

[0076] Then, the mitochondria in the living COS7 cells were fluorescently labeled, and the imaging method described in Example 3 was used to perform sub-pixel matching fluorescence and three-dimensional refractive index dual-modal imaging of the living COS7 cells, as shown in FIG. Figure 16 As shown, Figure 16 (a) shows the refractive index distribution (left) and mitochondrial fluorescence imaging (right) of a living COS7 cell, and, Figure 16 (b) and Figure 16 (c) presents Figure 16 (a) The refractive index distribution and mitochondrial fluorescence image of the white boxed area. This demonstrates that the present invention achieves sub-pixel-matched fluorescence and 3D refractive index dual-modality imaging using a single camera without any image processing. This further demonstrates the effectiveness of the present invention.

[0077] The present invention proposes a three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection, which have the following advantages: 1. It is suitable for oil immersion objective lenses and can obtain the three-dimensional refractive index distribution of the sample under test without specific labeling or three-dimensional scanning of the sample under test, thereby performing high-contrast and high-resolution three-dimensional refractive index imaging of the sample under test; 2. It realizes self-interference tomography based on the backscattered field for the first time, and combines it with quantitative phase contrast tomography based on the forward scattered field to achieve three-dimensional refractive index imaging over a wide spectral range, which is applicable to samples under test with different scattering degrees; 3. The speed and image quality of three-dimensional refractive index imaging are greatly improved by the multi-channel polarization detection technology of the polarization camera, the partially coherent scanning illumination of the light-emitting diode, and the phase modulation multiplexing strategy based on the spatial light modulator; 4. The optical structure of common-path interference realizes label-free three-dimensional imaging with excellent stability; 5. It can perform high-speed, high-quality, and highly stable three-dimensional label-free refractive index imaging of subcellular organelles in living cells or biological tissue samples under test; 6. By utilizing the selective spectroscopic principle of a dichroic mirror, it realizes sub-pixel-level matching fluorescence and three-dimensional refractive index dual-modality imaging with a single camera for the first time. Therefore, the three-dimensional imaging system and method based on hybrid scattered field and polarization phase contrast detection of the present invention can perform label-free, high-speed, high-quality, and highly stable in-situ three-dimensional refractive index detection of the sample to be tested, and has very good scalability in structure and function, and has great application value in fields such as industrial detection and life science research.

[0078] In the several embodiments provided herein, it should be understood that the apparatus and method disclosed herein can be implemented in other ways. For example, the apparatus embodiments described above are merely illustrative. For example, the module division is merely a logical functional division. In actual implementation, other division methods may be used. For example, multiple modules or components may be combined or integrated into another system, or some features may be ignored or not implemented.

[0079] In addition, the functional modules in various embodiments of the present invention may be integrated into a single processing module, each module may exist physically separately, or two or more modules may be integrated into a single module. The aforementioned integrated modules may be implemented in the form of hardware or hardware plus software functional modules.

[0080] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection, characterized in that: The invention comprises an epi-illumination module, a first thin lens (4), a non-polarizing beam splitter prism (5), a tube lens (6), a microscope objective lens (7), a second thin lens (9), a linear polarizer (10), a phase-type spatial light modulator (11), a third thin lens (12) and a polarization camera (13), wherein: The epi-illumination module is used to generate non-polarized and partially coherent divergent light in different directions at different times; The first thin lens (4) is used to collimate the divergent light and to make the collimated light beam incident on the non-polarizing beam splitter prism (5) at a preset inclination angle; the tube lens (6) and the microscope objective lens (7) are sequentially arranged in the reflection direction of the non-polarizing beam splitter prism (5); and the non-polarizing beam splitter prism (5) is used to reflect a portion of the collimated light beam to the tube lens (6); The back focal plane of the barrel lens (6) coincides with the back focal plane of the first thin lens (4), the front focal plane of the barrel lens (6) coincides with the back focal plane of the microscope objective lens (7), and the sample to be measured (8) is arranged at the front focal plane of the microscope objective lens (7) through a glass slide, wherein the collimated light beam passes through the barrel lens (6) and the microscope objective lens (7) and is sequentially irradiated onto the glass slide and the sample to be measured (8), and the glass slide generates mirror irradiation light, and the sample to be measured (8) generates a two-dimensional backscattering field; The second thin lens (9), the linear polarizer (10), the phase-type spatial light modulator (11), the third thin lens (12) and the polarization camera (13) are sequentially arranged on a side of the non-polarizing beam splitter prism (5) away from the tube lens (6); the front focal plane of the second thin lens (9) coincides with the back focal plane of the tube lens (6); the phase-type spatial light modulator (11) is arranged at the common focal plane of the second thin lens (9) and the third thin lens (12); a phase modulation pattern is provided on the phase-type spatial light modulator (11), which applies a phase modulation of 0.5π only to the mirror irradiation light, and applies a phase modulation of 1.5π to the two-dimensional backscattered field in other areas; The working surface of the polarization camera (13) is located at the back focal plane of the third thin lens (12).

2. The three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 1, characterized in that: The epi-illumination module comprises a first annular illuminator (1), a first industrial lens (2) and a first multimode optical fiber coupler (3), wherein: The first annular illuminator (1) comprises a plurality of light-emitting diodes arranged in an annular manner, wherein the plurality of light-emitting diodes are identical and can be lit in sequence to generate non-polarized and partially coherent oblique divergent light; The first industrial lens (2) is used to collect and scale the oblique divergent light and then couple it to the first multimode optical fiber coupler (3); The first multimode fiber coupler (3) comprises a plurality of identical multimode optical fibers, the input ports and output ports of the plurality of multimode optical fibers being evenly distributed on two circular rings to form an input ring and an output ring, respectively, and the axes of the input ring and the output ring coincide with the axis of the first industrial lens (2); the number of the multimode optical fibers is the same as the number of the light-emitting diodes, so that the first m The light emitted by the first LED is m Multimode optical fibers are used for coupling; The output ring of the first multimode optical fiber coupler (3) is located at the front focal plane of the first thin lens (4).

3. The three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 1, characterized in that: The two-dimensional backscattered field and specular illumination light generated by the sample to be measured (8) and the glass slide at the front focal plane of the microscope objective lens (7) are propagated to the rear focal plane thereof under the action of spatial Fourier transform of the microscope objective lens (7), wherein: The spectrum information of the two-dimensional backscattered field is distributed at the rear focal plane of the microscope objective lens (7) and is limited by the limited pupil aperture of the microscope objective lens (7). The mirror illumination light converges at the rear focal plane of the microscope objective lens (7). The mirror illumination light appears as a convergent light spot with a predetermined size at the rear focal plane of the microscope objective lens (7), and the convergent light spot has a predetermined lateral offset relative to the system optical axis.

4. The three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 1, characterized in that: The polarization direction of the linear polarizer (10) is 45 degrees to both the positive direction of the X-axis and the positive direction of the Y-axis, wherein the positive direction of the X-axis and the positive direction of the Y-axis are perpendicular to each other and parallel to the sample surface of the sample to be measured (8); the linear polarizer (10) is used to separate the two-dimensional backscattered field and the specular illumination light from the second thin lens (9) into linear polarized light along the X-axis direction and linear polarized light along the Y-axis direction with equal power; The phase-type spatial light modulator (11) performs phase modulation only on linearly polarized light along the X-axis direction; Each pixel of the polarization camera (13) consists of four sub-pixels, which are respectively used to detect linearly polarized light in the directions of 0°, 45°, 90° and 135°, so that one exposure of the polarization camera (13) can simultaneously generate four interference intensity maps.

5. The three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 1, characterized in that: The phase-type spatial light modulator (11) is provided with a phase modulation pattern based on a phase modulation multiplexing strategy, so that when any light-emitting diode in the first ring illuminator (1) emits light, the phase modulation pattern can apply a phase modulation of 0.5π to the corresponding mirror illumination light, and apply a phase modulation of 1.5π to the two-dimensional backscattered field in other spectral regions.

6. The three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 2, characterized in that: It also includes a transmission lighting module (14), which includes a second annular illuminator (14-1), a second industrial lens (14-2), a second multimode fiber coupler (14-3), a fourth thin lens (14-4), a fifth thin lens (14-5) and an illumination objective lens (14-6) arranged in sequence, wherein: The second annular illuminator (14-1) comprises a plurality of light-emitting diodes arranged in an annular manner, wherein the plurality of light-emitting diodes are identical and can be lit in sequence to generate non-polarized and partially coherent oblique divergent light; The plurality of light-emitting diodes arranged in an annular pattern on the second annular illuminator (14-1) are identical to the plurality of light-emitting diodes arranged in an annular pattern on the first annular illuminator (1) and have the same light-emitting spectrum; The second industrial lens (14-2) is used to collect and scale the oblique divergent light generated by the second ring illuminator (14-1) and couple the collected light to the second multimode fiber coupler (14-3). The second multimode fiber coupler (14-3) comprises a plurality of identical multimode optical fibers, the input ports and output ports of the plurality of multimode optical fibers being evenly distributed on two circular rings to form an input ring and an output ring, and the axes of the input ring and the output ring coincide with the axis of the second industrial lens (14-2); the number of the multimode optical fibers of the second multimode fiber coupler (14-3) is the same as the number of the light-emitting diodes of the second annular illuminator (14-1), so that the second multimode fiber coupler (14-3) is m The light emitted by the first LED is m Multimode optical fibers are used for coupling; And, from the m The divergent light emitted from the output port of the multimode optical fiber can be converged to the confocal plane of the fifth thin lens (14-5) and the illumination objective lens (14-6) after propagating through the confocal system composed of the fourth thin lens (14-4) and the fifth thin lens (14-5), and is obliquely irradiated onto the sample to be measured (8) located at the confocal plane of the illumination objective lens (14-6) and the microscope objective lens (7) in the form of a quasi-plane wave.

7. The three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 1, characterized in that: The three-dimensional imaging system comprises a fluorescent illumination module (15), a sixth thin lens (16), a dichroic mirror (17) and a filter (18), wherein: The emission surface of the fluorescent illumination module (15) is located at the front focal plane of the sixth thin lens (16), so that the fluorescent excitation light generated by the fluorescent illumination module (15) reaches the front focal plane of the microscope objective lens (7) after propagating through the confocal system composed of the sixth thin lens (16), the dichroic mirror (17) and the microscope objective lens (7), and performs specific fluorescence excitation on the sample to be measured (8) at the front focal plane of the microscope objective lens (7), thereby generating a fluorescent radiation signal; The phase-type spatial light modulator (11) loads a phase modulation pattern with a full area of ​​0π during the fluorescence imaging process and does not modulate the fluorescence radiation signal.

8. A three-dimensional imaging method based on mixed scattered field and polarization phase contrast detection, characterized in that: include: S1: lighting up each light-emitting diode of the first ring illuminator (1) of the three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to any one of claims 2 to 4 in sequence, and obtaining four first interference intensity images when each light-emitting diode is lit using a polarization camera (13) under epi-oblique illumination conditions; S2: Obtain the total spectrum distribution of the scattering potential of the sample to be tested based on the four first interference intensity images when each light-emitting diode is lit, and if the sample to be tested is a strong scattering sample, obtain the refractive index distribution of the sample to be tested using the total spectrum distribution of the scattering potential.

9. The three-dimensional imaging method based on hybrid scattered field and polarization phase contrast detection according to claim 8, characterized in that: Also includes: If the sample to be tested is a weakly scattering sample to be tested, each light-emitting diode of the second ring illuminator (14-1) in the three-dimensional imaging system based on hybrid scattered field and polarization phase contrast detection according to claim 5 is sequentially illuminated, and four second interference intensity images when each light-emitting diode is illuminated are obtained using a polarization camera (13) under transmissive oblique illumination conditions; The total transmission spectrum distribution of the scattering potential of the sample to be tested is obtained based on the four second interference intensity diagrams when each light-emitting diode is lit; the total incident spectrum distribution and the total transmission spectrum distribution are linearly superimposed to obtain the total spectrum distribution of the scattering potential of the sample to be tested, and the refractive index distribution of the sample to be tested is obtained using the total spectrum distribution.

10. The three-dimensional imaging method based on hybrid scattered field and polarization phase contrast detection according to claim 9, characterized in that: Before the S1, it also includes: Determine the wave vector generated by each light-emitting diode in the epi-illumination module on the sample surface, and determine the phase modulation pattern loaded on the phase-type spatial light modulator based on the phase modulation multiplexing strategy, so that all light-emitting diodes in the epi-illumination module share a phase modulation pattern; or Determine the wave vector generated by each light-emitting diode in the epi-illumination module on the sample surface and the wave vector generated by each light-emitting diode in the transmissive illumination module on the sample surface, so that the transverse wave vector generated by each light-emitting diode in the epi-illumination module on the sample surface and the transverse wave vector generated by each light-emitting diode in the transmissive illumination module on the sample surface are one-to-one and identical, and determine the phase modulation pattern loaded on the phase-type spatial light modulator based on the phase modulation multiplexing strategy, so that all light-emitting diodes in the epi-illumination module and all light-emitting diodes in the transmissive illumination module share a phase modulation pattern.

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