Optical diffraction tomography microscopy apparatus and method based on an integrated quantitative phase imaging camera

Abstract

The application discloses an optical diffraction tomography imaging device and method based on an integrated quantitative phase imaging camera, and mainly solves the technical problems of mechanical vibration, poor stability, low measurement precision, limited imaging field of view and the like of the current optical diffraction tomography device. The application realizes multi-angle rotary illumination without mechanical movement by loading a blazed phase grating with different directions and periods of pixel numbers on a reflective liquid crystal spatial light modulator; uses the integrated quantitative phase imaging camera to record the point array interference diagram of a non-marked transparent sample under different illumination angles, then processes the four-wave shearing interference point array diagram by using a Fourier transform method, and inversely calculates the light field complex amplitude distribution of the sample under different illumination angles; finally, based on the optical diffraction tomography principle, the three-dimensional refractive index distribution of the sample is quantitatively reproduced from the obtained multi-angle complex amplitude sequence, the micro three-dimensional structure distribution inside the transparent sample is observed without marking, and high-precision three-dimensional imaging is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of optical microscopic imaging technology, and particularly relates to an optical diffraction tomographic microscopic imaging device and method based on an integrated quantitative phase imaging camera. BACKGROUND

[0002] Optical diffraction tomography is a new optical label-free three-dimensional microscopic imaging technology, which can quantitatively observe the three-dimensional distribution of internal refractive index of a transparent sample. This technology can be used for long-time, non-invasive and high-resolution three-dimensional observation of living cells, and has been applied in the fields of immunology, hematology and cell biology.

[0003] According to the three-dimensional morphology and quantitative refractive index distribution of biological cells, important information such as the volume and dry weight of the target cells can be further obtained, meeting the needs of biomedical research. For example, Hayakawa et al. of the Japanese Autonomous Medical University observed the metabolic disorder phenomenon of red blood cells caused by Plasmodium falciparum infection by using this technology; Habaza et al. of the Tel Aviv University in Israel measured the refractive index distribution of white blood cells in response to immune action, providing important data support for the study of cell immune mechanism. In addition, the refractive index information inside the cells can also be used to monitor various physiological processes of the cells or to screen target cells. For example, Sung et al. of the Massachusetts Institute of Technology in the United States observed the different processes of biological cell mitosis by measuring the refractive index of chromosomes; Hsu et al. of the University of Taiwan in China screened cancer cells by using the characteristic that the refractive index of cancer cells is higher than that of normal cells.

[0004] The current optical diffraction tomographic device is usually composed by adding a two-dimensional galvanometer to a Mach-Zehnder interference-based digital holographic microscopic device. The two-dimensional galvanometer is used to change the illumination angle, and then the digital holographic technology is used to obtain the complex amplitude of the sample under the corresponding illumination angle. However, the nonlinear response characteristics of the two-dimensional galvanometer may cause the actual illumination angle to deviate from the expected illumination angle, thereby affecting the observation accuracy. In addition, the Mach-Zehnder interference-based digital holographic microscopic device is easily affected by external environmental disturbances and mechanical vibrations during the operation of the two-dimensional galvanometer, resulting in measurement errors, and the cumulative effect of measurement errors under different illumination angles will seriously affect the accuracy of refractive index measurement.

[0005] To solve the above problems, the Park team of the Korea Advanced Science and Technology Research Institute uses a digital micromirror device instead of a two-dimensional galvanometer to construct an optical diffraction tomography device, thereby effectively avoiding mechanical vibration caused by the two-dimensional galvanometer. However, the device still uses a Mach-Zehnder interference structure, which is susceptible to environmental disturbances, thereby affecting the stability of the device. Based on this, the Sung team of the National Taiwan University and the Truiak team of the Warsaw University of Technology respectively use gratings and spatial filtering devices to construct a phase imaging device with a common path of an object, which can effectively improve the stability of the device, but also derives the problem of limited imaging field of view. SUMMARY

[0006] The present application aims at the technical problems of mechanical vibration, poor stability, low measurement accuracy, and limited imaging field of view of the current optical diffraction tomography device, and provides an optical diffraction tomography imaging device and method based on an integrated quantitative phase imaging camera.

[0007] To solve the above problems, the technical solutions of the present application are as follows:

[0008] An optical diffraction tomography imaging device based on an integrated quantitative phase imaging camera is used for optical diffraction tomography imaging of a sample to be measured, and is characterized in that:

[0009] The device comprises a rotating illumination unit, a microscopic imaging unit, and a recording unit.

[0010] The rotating illumination unit comprises a laser, and a beam expansion collimation system, an optical modulation system, and a telescope system arranged in sequence along the outgoing light of the laser.

[0011] The beam expansion collimation system is used to expand and collimate the outgoing light into a plane wave beam.

[0012] The optical modulation system comprises a triangular reflecting prism and a spatial light modulator. The triangular reflecting prism is used to turn the transmission light path of the plane wave beam, and the cross section thereof is an isosceles triangle. One side of the triangular reflecting prism close to the beam expansion collimation system is defined as a first reflecting surface, and the other side opposite to the first reflecting surface is defined as a second reflecting surface.

[0013] The spatial light modulator is a reflective liquid crystal spatial light modulator, which is located above the first reflecting surface and the second reflecting surface and has a facet close to the first reflecting surface and the second reflecting surface loaded with a blazed phase grating with different directions and periodic pixel numbers, which is used to modulate the transmission angle of the plane wave beam. The plane wave beam reaches the light entrance end of the telescope system in sequence through the first reflecting surface, the spatial light modulator, and the second reflecting surface.

[0014] The telescope system is used to expand the scanning angle range of the modulated plane wave beam.

[0015] The microscope imaging unit comprises a stage and a microscope imaging system; the lower surface of the stage is close to the light exit end of the telescope system, and the upper surface is used for placing the sample to be tested, so that the sample to be tested is located on the front focal plane of the microscope imaging system; the stage is used for cutting into the light path when testing the sample to be tested and cutting out of the light path when testing the background; and the microscope imaging system is used for acquiring information of the sample to be tested.

[0016] The recording unit is an integrated quantitative phase imaging camera, which is used for recording and calculating the imaging information of the sample to be tested and outputting the image information of the sample to be tested.

[0017] Further, the beam expansion collimation system comprises a first lens and a second lens arranged in sequence along the exit light of the laser, wherein the first lens is a beam expansion mirror, which is arranged close to the laser.

[0018] Further, the telescope system comprises a third lens and an illumination objective arranged in sequence along the light path, wherein the third lens is close to the second reflecting surface, the front focal plane coincides with the plane on which the spatial light modulator is loaded with the blazed phase grating, and the back focal plane coincides with the back focal plane of the illumination objective.

[0019] Further, a reflecting mirror is arranged between the second reflecting surface of the triangular reflecting prism and the third lens, which is used for turning the light path.

[0020] Further, the microscope imaging system comprises an imaging objective and a sleeve lens arranged in sequence along the light path; the front focal plane of the imaging objective coincides with the front focal plane of the illumination objective, the sample to be tested placed on the stage is located at the focal plane coinciding with the illumination objective and the imaging objective; the front focal plane of the sleeve lens coincides with the back focal plane of the imaging objective (10); and the recording unit is located at the back focal plane of the sleeve lens.

[0021] Meanwhile, the application also provides an optical diffraction tomography microscopic imaging method based on the integrated quantitative phase imaging camera, which is characterized in that the optical diffraction tomography microscopic imaging device based on the integrated quantitative phase imaging camera is used, and the method comprises the following steps:

[0022]

S1

[0023]

S2

[0024] The microscopic imaging system receives the illumination light carrying sample information, and obtains the imaging information of the sample to be measured, and then transmits the sample light field interference point array I(x, y) of the sample to be measured under a series of different illumination angles to the recording unit, wherein (x, y) represents the coordinates of the pixel points of the recording unit.

[0025]

S4

[0026]

S5

[0027]

S6

S3

S5

[0028] Further, step

S6

[0029] 【6.1】The recording unit obtains the sample light field complex amplitude u(x, y) under different illumination angles by inversely calculating the sample light field interference point array I(x, y) obtained in step

S3

[0030] 【6.2】The background light field complex amplitude u0(x, y) under different illumination angles is obtained by calculating the background light field interference point array I0(x, y) obtained in step

S5

[0031] 【6.3】Based on the sample light field complex amplitude u(x, y) obtained in step 【6.1】 and the background light field complex amplitude u0(x, y) of the sample to be measured obtained in step 【6.2】, the three-dimensional refractive index distribution of the sample to be measured is reconstructed, and the imaging of the sample to be measured is realized.

[0032] Further, step 【6.1】 comprises the following steps:

[0033] 【6.1.1】 Fourier transforming the sample light field interference pattern I(x, y) at any one illumination angle obtained in step

S3

[0034] 【6.1.2】 On the basis of step 【6.1.1】, at the +1-order spectrum in the xy two orthogonal directions, a filter window function G1 is respectively applied to filter the +1-order spectrum in the x direction, and a filter window function G2 is respectively applied to filter the +1-order spectrum in the y direction, and then the complex phase angle of the filtered +1-order spectrum is obtained by performing inverse Fourier transformation, so as to obtain the phase gradient in the x direction and the phase gradient in the y direction

[0035]

[0036]

[0037] wherein η is a calculation coefficient determined by the basic parameters of the recording unit, and arg represents the complex phase angle;

[0038] 【6.1.3】 Two-dimensional gradient integration is respectively performed on the phase gradient in the x direction and the phase gradient in the y direction obtained in step 【6.1.2】 to obtain the phase distribution of the sample to be measured at the arbitrary illumination angle

[0039]

[0040] wherein (x0, y0) is the coordinate of the starting point of the path integral;

[0041] 【6.1.4】 The amplitude distribution A(x, y) obtained in step 【6.1.1】 and the phase distribution obtained in step 【6.1.3】 are substituted into the formula , so as to obtain the complex amplitude u(x, y) of the sample light field of the sample to be measured at the arbitrary illumination angle;

[0042] 【6.1.5】 Steps 【6.1.1】 to 【6.1.4】 are repeated until the complex amplitude of the sample light field of the sample to be measured at all illumination angles is obtained.

[0043] Further, step 【6.3】 comprises the following steps:

[0044] 【6.3.1】According to Rytov approximation, the scattering field u of the sample to be measured at different illumination angles is calculated s (x,y):

[0045]

[0046] 【6.3.2】The two-dimensional Fourier transform of the scattering field u s (x,y) at different illumination angles is carried out to obtain the scattering field spectrum, and the scattering field spectrum is filled into the three-dimensional spectrum space to obtain the three-dimensional spectrum F(k) of the sample to be measured:

[0047]

[0048] U s (k i,x ,k i,y ) is the two-dimensional Fourier transform of u s (x,y);k=(k x ,k y ,k z ) is the three-dimensional spectrum coordinate;k i =(k i,x ,k i,y ,k i,z ) is the illumination light direction vector, and where k0 is the illumination light wave vector, k0=2π / λ, λ is the wavelength of the illumination light;n m is the refractive index of the medium surrounding the sample to be measured;

[0049] 【6.3.3】The three-dimensional spectrum F(k) of the sample to be measured is inversely Fourier transformed to obtain the scattering potential function f(r) of the sample to be measured;

[0050] 【6.3.4】According to the conversion relationship between the scattering potential function f(r) of the sample to be measured and the refractive index n(r) The three-dimensional refractive index distribution information of the sample to be measured is calculated to realize the imaging of the sample to be measured.

[0051] Further, in step

S2

[0052]

[0053] where λ is the wavelength of the illumination light, m is the number of pixels in one period, p is the pixel size of the spatial light modulator, and + or - depends on the slope direction of the linear phase.

[0054] Compared with the prior art, the application has the following advantages:

[0055] 1. The optical diffraction tomography imaging device based on the integrated quantitative phase imaging camera provided by the application uses the integrated quantitative phase imaging camera to obtain the complex amplitude distribution of a sample under different illumination angles, without a reference light, and has the advantages of simple overall structure, strong anti-environmental disturbance ability and high stability.

[0056] 2. The optical diffraction tomography imaging device based on the integrated quantitative phase imaging camera provided by the application uses a reflective liquid crystal spatial light modulator to realize rotary illumination, without any mechanical vibration, and can realize stable recording, laying a foundation for subsequent high-precision measurement.

[0057] 3. The optical diffraction tomography imaging method based on the integrated quantitative phase imaging camera provided by the application can observe the internal three-dimensional refractive index distribution of a label-free sample, has a simple operation process, and has high measurement precision and imaging quality. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 It is a structural schematic diagram of the optical diffraction tomography imaging device based on the integrated quantitative phase imaging camera provided by the application.

[0059] Figure 2 It is a schematic diagram of the blazed phase grating structure loaded on the spatial light modulator and its working principle in the optical diffraction tomography imaging device based on the integrated quantitative phase imaging camera provided by the application, wherein (a) is the schematic diagram of the blazed phase grating structure loaded on the spatial light modulator and its working principle when θ x is a negative included angle; (b) is the schematic diagram of the blazed phase grating structure loaded on the spatial light modulator and its working principle when θ x is a positive included angle.

[0060] Figure 3 It is a schematic diagram of the principle and flow of reconstructing the intensity image and the phase image from the dot array interference pattern of one phase resolution plate in the optical diffraction tomography imaging method based on the integrated quantitative phase imaging camera provided by the application.

[0061] Figure 4 It is a schematic diagram of the blazed grating phase image loaded in the optical diffraction tomography imaging method based on the integrated quantitative phase imaging camera provided by the application and the corresponding illumination angle distribution.

[0062] Figure 5Figure (a) is a schematic diagram of the three-dimensional spectrum of the scattering field under different illumination angles; and Figure (b) is a schematic diagram of the completely filled three-dimensional spectrum of the scattering field under all illumination angles.

[0063] Figure 6 Figure (a) is a three-dimensional refractive index distribution map of the polystyrene microspheres with a diameter of 7 μm; Figure (b) is a refractive index distribution map of the polystyrene microspheres with a diameter of 7 μm in the x-y profile and the y-z profile; and Figure (c) is a comparison map of the measured value and the actual value of the polystyrene microspheres with a diameter of 7 μm in the x-y profile and the y-z profile.

[0064] Figure 7 Figure (a) is a three-dimensional refractive index distribution map of the polystyrene microspheres with a diameter of 7 μm; Figure (b) is a refractive index distribution map of the polystyrene microspheres with a diameter of 7 μm in the x-y profile and the y-z profile; and Figure (c) is a comparison map of the measured value and the actual value of the polystyrene microspheres with a diameter of 7 μm in the x-y profile and the y-z profile.

[0065] The reference signs are as follows:

[0066] 1-laser; 2-first lens; 3-second lens; 4-triangle reflecting prism; 41-first reflecting surface; 42-second reflecting surface; 5-space light modulator; 6-reflecting mirror; 7-third lens; 8-illumination objective; 9-objective table; 10-imaging objective; 11-sleeve lens; and 12-integrated quantitative phase imaging camera. DETAILED DESCRIPTION

[0067] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below with reference to the accompanying drawings. The described embodiments are part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the protection scope of the present application.

[0068] In the following description, many specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in other manners different from those described herein without departing from the scope of the present application. Those skilled in the art can make similar extensions without departing from the spirit of the present application, and therefore the present application is not limited to the specific embodiments disclosed below.

[0069] As Figure 1As shown, the embodiment provides an optical diffraction tomography imaging device based on an integrated quantitative phase imaging camera, mainly used for optical diffraction tomography imaging of a sample to be measured. The imaging device comprises a rotating illumination unit, a microscopic imaging unit and a recording unit. The rotating illumination unit comprises a laser 1, a beam expansion collimation system, an optical modulation system and a telescope system arranged in sequence along the outgoing light of the laser 1. The model of the laser 1 is MLL-U-SM-532, and the laser wavelength is 532 nm.

[0070] The beam expansion collimation system is used to expand and collimate the outgoing light into a plane wave beam. The beam expansion collimation system comprises a first lens 2 and a second lens 3 arranged in sequence along the outgoing light. The first lens 2 is a beam expansion mirror arranged close to the laser 1. The second lens 3 is a collimation mirror. The focal lengths of the first lens 2 and the second lens 3 are 7.5 mm and 200 mm, respectively.

[0071] The optical modulation system comprises a triangular reflecting prism 4 and a spatial light modulator 5. The triangular reflecting prism 4 is used to turn the transmission path of the plane wave beam. The cross section of the triangular reflecting prism 4 is an isosceles triangle. In this embodiment, a triangular reflecting prism 4 with a top angle of 96° is selected. Here, one side of the triangular reflecting prism 4 close to the beam expansion collimation system is defined as a first reflecting surface 41, and the other side opposite to the first reflecting surface 41 is defined as a second reflecting surface 42.

[0072] The spatial light modulator 5 is a reflective liquid crystal spatial light modulator. The spatial light modulator 5 is located above the first reflecting surface 41 and the second reflecting surface 42, and one side of the spatial light modulator 5 close to the first reflecting surface 41 and the second reflecting surface 42 is loaded with a blazed phase grating for modulating the transmission angle of the plane wave beam. The plane wave beam passes through the first reflecting surface 41, the spatial light modulator 5 and the second reflecting surface 42 in sequence and then reaches the light entrance end of the telescope system.

[0073] The telescope system is used to expand the scanning angle range of the modulated plane wave beam. The telescope system comprises a third lens 7 and an illumination objective 8 arranged in sequence along the light path. The third lens 7 is close to the second reflecting surface 42, and the front focal plane of the third lens 7 coincides with the plane on which the panel of the spatial light modulator 5 loaded with the blazed phase grating is located. The rear focal plane of the third lens 7 coincides with the rear focal plane of the illumination objective 8. The focal length of the third lens 7 is 300 mm.

[0074] In order to shorten the optical path and make the overall structure of the imaging device more compact, the embodiment further comprises a reflecting mirror 6 located between the second reflecting surface 42 of the triangular reflecting prism 4 and the third lens 7 for turning the optical path.

[0075] The microscopic imaging unit comprises a stage 9 and a microscopic imaging system; the microscopic imaging system is used to acquire information of the sample to be measured, and comprises an imaging objective 10 and a sleeve lens 11 arranged in sequence along an optical path; the front focal plane of the imaging objective 10 coincides with the front focal plane of the illumination objective 8, the lower surface of the stage 9 is close to the light exit end of the telescope system, the upper surface of the stage 9 is used to place the sample to be measured, and the sample to be measured is located at the focal plane coinciding with the illumination objective 8 and the imaging objective 10. The focal length of the sleeve lens 11 is 200 mm, the front focal plane of the sleeve lens 11 coincides with the rear focal plane of the imaging objective 10; a recording unit is located at the rear focal plane of the sleeve lens 11, and is used to acquire information obtained by the microscopic imaging system.

[0076] In the embodiment, the illumination objective 8 and the imaging objective 10 are both water objectives, and the magnification and the numerical aperture are both 60x and 1.27.

[0077] The recording unit is an integrated quantitative phase imaging camera 12, which is used to record and calculate the imaging information of the sample to be measured, and output the image information of the sample to be measured.

[0078] In addition, the embodiment also provides an optical diffraction tomographic microscopic imaging method based on an integrated quantitative phase imaging camera, comprising the following steps:

[0079]

S1

[0080]

S2

[0081] Specifically, as shown in the figure, Figure 2 the blazed phase grating is loaded in the x direction of the reflective liquid crystal spatial light modulator, the period is mp (m is the number of pixels in one period, and p is the pixel size of the reflective liquid crystal spatial light modulator), each period has a grating structure with a linear phase change of 0-2π, the blazed phase grating diffracts the light beam incident on the reflective liquid crystal spatial light modulator, and the included angle θ x According to the formula:

[0082]

[0083] Wherein, λ is the wavelength of the illumination light, + or - depends on the slope direction of the linear phase.

[0084] The above formula shows that the direction of the illumination light beam exiting the reflective liquid crystal spatial light modulator can be changed by adjusting the pixel number m of the grating period and the phase slope direction. Therefore, by sequentially changing the pixel number of the period and the phase slope direction of the blazed phase grating loaded on the reflective liquid crystal spatial light modulator in different directions in the xy plane, multi-angle rotary illumination of the sample to be measured on the xy two-dimensional plane can be realized.

[0085] The exiting light diffracted by the reflective liquid crystal spatial light modulator then reaches the telescope system through the second reflecting surface 42 of the triangular reflecting prism 4 and the mirror 6. Since the range of the rotary angle of the illumination light modulated by the above method is limited, a 4f telescope system composed of the third lens 7 and the illumination objective lens 8 is required to enlarge the included angle between the illumination light and the optical axis, so as to meet the requirement of the tilt angle. Finally, the exiting light is transmitted to the sample to be measured at different angles, forming the illumination light carrying the sample information.

[0086]

S3

[0087] As shown in Figure 4 In order to collect sufficient data, 73 tilt illumination angles are set in the embodiment, which correspond to the positions at the back focal plane BFP of the illumination objective lens 8, wherein the polar angle of the illumination angle about the z axis is θ, and the azimuth angle about the x axis is φ.

[0088]

S4

S2

S2

[0089]

S5

[0090]

S6

S3

S5

[0091] 【6.1】The recording unit inversely obtains the complex amplitude u(x, y) of the sample light field under different illumination angles by the four-wave shearing interferogram processing method from the sample light field interference pattern I(x, y) obtained in step

S3

[0092] 【6.1.1】The Fourier transform (FT) is performed on the sample light field interference pattern I(x, y) under any one of the illumination angles obtained in step

S3

[0093] A(x, y) = |IFT{G0·FT[I(x, y)]}| (2)

[0094] 【6.1.2】On the basis of step 【6.1.1】, at the +1 order frequency spectrum in the xy two orthogonal directions, the filter window function G1 is applied to filter out the +1 order frequency spectrum in the x direction, the filter window function G2 is applied to filter out the +1 order frequency spectrum in the y direction, and the complex phase angle (arg) is taken after the filtered +1 order frequency spectrum is subjected to inverse Fourier transform, and then the phase gradient in the x direction and the phase gradient in the y direction

[0095]

[0096] wherein η is a calculation coefficient determined by the basic parameters of the integrated quantitative phase imaging camera 12.

[0097] 【6.1.3】The two-dimensional gradient integration is performed on the phase gradient in the x direction and the phase gradient in the y direction obtained in step 【6.1.2】 to obtain the phase distribution

[0098]

[0099] wherein (x0, y0) is the coordinate of the starting point of the path integration.

[0100] 【6.1.4】The amplitude distribution A(x, y) obtained in step 【6.1.1】 and the phase distribution obtained in step 【6.1.3】 are substituted into the formula The sample light field complex amplitude u(x, y) of the sample under test at the arbitrary illumination angle is obtained, wherein exp represents the exponential function with e as the base, and i represents the imaginary symbol.

[0101] 【6.1.5】Repeat steps 【6.1.1】 to 【6.1.4】 until the sample light field complex amplitude of the sample under test at all illumination angles is obtained.

[0102] The process of filtering the reconstructed light field complex amplitude from the spectrum of the lattice interference pattern is shown in Figure 3 .

[0103] 【6.2】According to the method of step 【6.1】, the background light field interference lattice pattern I0(x, y) obtained in step

S5

[0104] 【6.3】Based on the sample light field complex amplitude u(x, y) obtained in step 【6.1】 and the background light field complex amplitude u0(x, y) of the sample under test obtained in step 【6.2】, the three-dimensional refractive index distribution of the sample under test is reconstructed, and the imaging of the sample under test is realized.

[0105] 【6.3.1】According to the Rytov approximation, the scattering field u s (x, y) of the sample under test under different illumination angles is calculated:

[0106]

[0107] 【6.3.2】The two-dimensional Fourier transform of the scattering field u s (x, y) under different illumination angles is performed to obtain the scattering field spectrum, and all the scattering field spectra under different illumination angles are filled into the three-dimensional spectrum space through the following relationship to obtain the three-dimensional spectrum F(k) of the sample under test:

[0108]

[0109] In the formula, U s (k i,x ,k i,y ) is the two-dimensional Fourier transform of u s (x, y); k = (k x ,k y ,k z ) is the three-dimensional spectrum coordinate; k i = (k i,x ,k i,y ,k i,z ) is the illumination light direction vector, and wherein k0 is the illumination light wave vector, k0 = 2π / λ, and λ is the wavelength of the illumination light; n mis the refractive index of the medium surrounding the sample.

[0110] 【6.3.3】The three-dimensional spectrum F(k) of the sample to be tested is subjected to inverse Fourier transform to obtain the scattering potential function f(r) of the sample to be tested, where r=(x,y,z), which are the three-dimensional spatial coordinates.

[0111] The process of filling the three-dimensional spectrum F(k) of the sample according to formula (6) is as follows: Figure 5 As shown. Figure 5 As shown in (a), u at each illumination angle s A two-dimensional spectrum of (x,y) can be filled onto a spherical shell of a three-dimensional spectrum, with the position of the shell varying depending on the illumination angle. The range of three-dimensional spectra that the complex amplitude can fill at all illumination angles is as follows: Figure 5 As shown in (b), the three-dimensional spectrum has a range of 4n in the transverse and axial directions. m sinα / λ and 2n m ·(1-cosα) / λα is the collection angle of the imaging objective lens 10, NA=n m ·sinα.

[0112] 【6.3.4】Based on the conversion relationship between the scattering potential function f(r) and the refractive index n(r) of the sample to be tested The three-dimensional refractive index distribution information n(r) of the sample under test is calculated, and the sample under test is imaged.

[0113] This invention uses an integrated quantitative phase imaging camera to measure the complex amplitude information of the scattering field of unmarked transparent or translucent samples under different illumination angles. Then, based on the principle of optical diffraction tomography, the three-dimensional refractive index inside the sample is reproduced from the obtained multi-angle complex amplitude sequence, and the microscopic three-dimensional structure distribution inside the transparent sample is observed without a label.

[0114] To verify the feasibility of this method, a 7 μm diameter polystyrene microsphere with a refractive index of 1.598 at 532 nm and a refractive index of 1.560 at 532 nm, impregnated with UV adhesive, was used as the sample. The reconstructed three-dimensional refractive index distribution of the polystyrene microsphere is shown below. Figure 6 As shown in (a), the refractive index of the microsphere in the xy section marked by S1 and the yz section marked by S2 is as follows: Figure 6 As shown in (b), Figure 6 As shown in (c), the dashed black lines “L1” and “L2” at the center of the two cross sections are analyzed. The measured value of the refractive index of the cross section center is consistent with the actual value of the dashed line.

[0115] To verify the imaging ability of the method on biological samples, three-dimensional refractive index measurement of a Chlorella prototecoides colony was performed. Chlorella prototecoides is a colony usually consisting of 16, 32 or 64 Chlorella-type cells arranged in a hollow sphere in a glycoprotein matrix. The three-dimensional refractive index measurement results of Chlorella prototecoides are shown in Figure 7 Fig. 3. Figure 7 From Fig. 3(a), it can be seen that Chlorella prototecoides is surrounded by a glycoprotein matrix with a refractive index slightly higher than water, with a refractive index of 1.342 to 1.350, and the center is a Chlorella-type cell with a higher refractive index, with a refractive index of 1.360 to 1.375. Figure 7 Fig. 3(b) is a refractive index distribution of three x-y sections "S1", "S2" and "S3" in Fig. 3(a). According to the figure, the number distribution of cells at different axial positions can be clearly distinguished, which proves the chromatographic imaging ability of the method. Figure 7 Fig. 3(b) is a refractive index distribution of three x-y sections "S1", "S2" and "S3" in Fig. 3(a). According to the figure, the number distribution of cells at different axial positions can be clearly distinguished, which proves the chromatographic imaging ability of the method.

[0116] In summary, the optical diffraction tomographic microscope device and method based on the integrated quantitative phase imaging camera can accurately obtain the three-dimensional refractive index distribution inside the biological sample, and has great application potential in the field of biological imaging.

[0117] The present application can realize the measurement of the three-dimensional refractive index distribution inside the transparent sample without mechanical scanning and reference light, and has the advantages of high stability, high phase measurement accuracy and convenient use.

Claims

1. An optical diffraction tomography imaging device based on an integrated quantitative phase imaging camera, for performing optical diffraction tomography imaging on a sample to be measured, characterized in that: it comprises a rotating illumination unit, a microscopic imaging unit and a recording unit; the rotating illumination unit comprises a laser (1), and a beam expanding collimating system, an optical modulation system and a telescope system arranged in sequence along the outgoing light of the laser (1); the beam expanding collimating system is used to expand and collimate the outgoing light into a plane wave beam; the optical modulation system comprises a triangular reflecting prism (4) and a spatial light modulator (5); the triangular reflecting prism (4) is used to turn the transmission light path of the plane wave beam, and its cross section is an isosceles triangle; one side of the triangular reflecting prism (4) close to the beam expanding collimating system is defined as a first reflecting surface (41), and the other side opposite to the first reflecting surface (41) is defined as a second reflecting surface (42); the spatial light modulator (5) is a reflective liquid crystal spatial light modulator, which is located above the first reflecting surface (41) and the second reflecting surface (42), and one side close to the first reflecting surface (41) and the second reflecting surface (42) is loaded with a blazed phase grating with different directions and periodic pixel numbers, which is used to modulate the transmission angle of the plane wave beam; the plane wave beam passes through the first reflecting surface (41), the spatial light modulator (5) and the second reflecting surface (42) in sequence, and then reaches the light entrance end of the telescope system; the telescope system is used to expand the scanning angle range of the modulated plane wave beam; the microscopic imaging unit comprises an object table (9) and a microscopic imaging system; the lower surface of the object table (9) is close to the light exit end of the telescope system, and the upper surface is used to place the sample to be measured so that the sample to be measured is located on the front focal plane of the microscopic imaging system; the object table (9) is used to cut into the light path when testing the sample to be measured, and cut out of the light path when testing the background; the microscopic imaging system is used to acquire the information of the sample to be measured; the recording unit is an integrated quantitative phase imaging camera (12), which is used to record and calculate the imaging information of the sample to be measured, and output the image information of the sample to be measured.

2. The optical diffraction tomography imaging device based on an integrated quantitative phase imaging camera according to claim 1, characterized in that: the beam expanding collimating system comprises a first lens (2) and a second lens (3) arranged in sequence along the outgoing light of the laser (1), wherein the first lens (2) is an expanding mirror, which is arranged close to the laser (1); and the second lens (3) is a collimating mirror.

3. The optical diffraction tomography imaging device based on an integrated quantitative phase imaging camera according to claim 2, characterized in that: the telescope system comprises a third lens (7) and an illumination objective lens (8) arranged in sequence along the light path, wherein the third lens (7) is close to the second reflecting surface (42), and the front focal plane coincides with the plane on which the blazed phase grating is loaded on one side of the spatial light modulator (5), and the rear focal plane coincides with the rear focal plane of the illumination objective lens (8).

4. The optical diffraction tomography imaging device based on an integrated quantitative phase imaging camera according to claim 3, characterized in that: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ A reflecting mirror (6) is arranged between the second reflecting surface (42) of the triangular reflecting prism (4) and the third lens (7) to change the light path.

5. The optical diffraction tomography imaging device based on the integrated quantitative phase imaging camera according to any one of claims 1 to 4, characterized in that: The microscopic imaging system comprises an imaging objective lens (10) and a sleeve lens (11) arranged in sequence along the light path; the front focal plane of the imaging objective lens (10) coincides with the front focal plane of the illumination objective lens (8), and the sample to be measured placed on the object table (9) is located at the focal plane coinciding with the illumination objective lens (8) and the imaging objective lens (10); the front focal plane of the sleeve lens (11) coincides with the back focal plane of the imaging objective lens (10); and the recording unit is located at the back focal plane of the sleeve lens (11).

6. An optical diffraction tomographic imaging method based on an integrated quantitative phase imaging camera, characterized in that, The optical diffraction tomography imaging device based on the integrated quantitative phase imaging camera according to any one of claims 1 to 5 comprises the following steps: 【S1】placing the sample to be measured on the object table (9) and adjusting the object table (9) so that the sample to be measured is located on the front focal plane of the microscopic imaging system; 【S2】the light emitted by the laser (1) is transmitted to the sample to be measured at different angles after passing through the beam expansion and collimation system, the first reflecting surface (41), the spatial light modulator (5), the second reflecting surface (42) and the telescope system, forming the illumination light carrying the sample information; 【S3】the microscopic imaging system receives the illumination light carrying the sample information and obtains the imaging information of the sample to be measured, which is then transmitted to the recording unit to record a series of sample light field interference point array graphs I(x, y) of the sample to be measured under different illumination angles, wherein (x, y) represents the coordinates of the pixel points of the recording unit; 【S4】moving the sample to be measured and the object table (9) out, and transmitting the light emitted by the laser (1) to the microscopic imaging system at different angles after passing through the beam expansion and collimation system, the first reflecting surface (41), the spatial light modulator (5), the second reflecting surface (42) and the telescope system; 【S5】the microscopic imaging system obtains the light information emitted at different angles and transmits it to the recording unit, which records a series of background light field interference point array graphs I0(x, y) of the sample to be measured under different illumination angles; 【S6】the recording unit calculates the sample light field interference point array graph I(x, y) obtained in step 【S3】 and the background light field interference point array graph I0(x, y) obtained in step 【S5】 respectively to obtain the three-dimensional refractive index distribution of the sample to be measured, thereby realizing the imaging of the sample to be measured.

7. The method of optical diffractometry tomographic imaging based on integrated quantitative phase imaging camera according to claim 6, wherein, Step 【S6】 comprises the following steps: 【6.1】the recording unit inversely obtains the sample light field complex amplitude u(x, y) under different illumination angles by processing the sample light field interference point array graph I(x, y) obtained in step 【S3】 through the four-wave shearing interference point array processing method; 【6.2】calculating the background light field interference point array graph I0(x, y) obtained in step 【S5】 according to the method of step 【6.1】 to obtain the background light field complex amplitude u0(x, y) under different illumination angles; and 【6.3】calculating the three-dimensional refractive index distribution of the sample to be measured by using the sample light field complex amplitude u(x, y) and the background light field complex amplitude u0(x, y) obtained in steps 【6.1】 and 【6.2】 respectively. 【6.3】Based on the sample light field complex amplitude u(x, y) obtained in step 【6.1】 and the background light field complex amplitude u0(x, y) of the sample to be measured obtained in step 【6.2】, the three-dimensional refractive index distribution of the sample to be measured is reconstructed, and the imaging of the sample to be measured is realized.

8. The method of optical diffractometry tomographic imaging based on integrated quantitative phase imaging camera according to claim 7, wherein, Step 【6.1】 comprises the following steps: 【6.1.1】 Fourier transform is performed on the sample light field interference point array I(x, y) of any one illumination angle obtained in step 【S3】 to obtain a frequency spectrum of the sample to be measured, a preset zero frequency filter window function G0 is used to filter out the zero-order spectrum located in the center of the frequency spectrum, and inverse Fourier transform is performed on the zero-order spectrum to obtain an intensity distribution of the sample to be measured at the arbitrary illumination angle, wherein the amplitude distribution in the intensity distribution is A(x, y), and A(x, y) = |IFT{G0·FT[I(x, y)]}|; 【6.1.2】On the basis of step 【6.1.1】, at the +1 order spectrum in the xy two orthogonal directions, respectively, a filter window function G1 is applied to filter out the +1 order spectrum in the x direction, a filter window function G2 is applied to filter out the +1 order spectrum in the y direction, and the complex phase angle is obtained after inverse Fourier transform of the filtered +1 order spectrum, thereby obtaining the phase gradient of the sample to be measured in the x direction at the arbitrary illumination angle and the phase gradient in the y direction Wherein, η is a calculation coefficient determined by the basic parameters of the recording unit, and arg represents a complex phase angle; 【6.1.3】performing two-dimensional gradient integration on the phase gradients in x direction and y direction respectively obtained in step 【6.1.2】 to obtain the phase distribution of the sample under test at this arbitrary illumination angle In the formula, (x0, y0) is the coordinate of the starting point of the path integral; 【6.1.4】obtaining the phase distribution Substituting the formula in the formula, the complex amplitude u(x, y) of the sample light field of the sample to be measured at the arbitrary illumination angle is obtained. 【6.1.5】 Repeat steps 【6.1.1】 to 【6.1.4】 until the sample light field complex amplitude of the sample to be measured at all illumination angles is obtained.

9. The method of optical diffractometry tomographic imaging based on integrated quantitative phase imaging camera according to claim 8, wherein, Step 【6.3】 comprises the following steps: [6.3.1] According to Rytov approximation, the scattering field u of the sample under test at different illumination angles is calculated s (x, y): 【6.3.2】Scattered field u at different illumination angles s (x,y) is two-dimensional Fourier transformed to obtain the scattered field spectrum, and the scattered field spectrum is filled into a three-dimensional spectrum space to obtain the three-dimensional spectrum F(k) of the sample under test: where U s (k i,x ,k i,y ) is a two-dimensional Fourier transform of u s (x,y); k = (k x ,k y ,k z ) is a three-dimensional frequency coordinate; k i = (k i,x ,k i,y ,k i,z ) is an illumination light direction vector, and where k0 is an illumination light wave vector, k0 = 2π / λ, λ is a wavelength of the illumination light; n m is a refractive index of a surrounding medium of the sample under test. 【6.3.3】 Fourier inverse transform is performed on the three-dimensional frequency spectrum F(k) of the sample to be measured to obtain a scattering potential function f(r) of the sample to be measured; 【6.3.4】According to the conversion relationship between the scattering potential function f(r) of the sample to be measured and the refractive index n(r) The three-dimensional refractive index distribution information of the sample to be measured is calculated, and the imaging of the sample to be measured is realized.

10. The optical diffraction tomographic imaging method based on the integrated quantitative phase imaging camera according to claim 8, characterized in that: In step S2, the outgoing light is transmitted to the sample to be measured at different angles by loading different direction and period pixel number of blazed phase grating on the spatial light modulator (5); the included angle θ between the outgoing light and the light beam incident on the spatial light modulator (5) x According to the formula: Wherein, λ is the wavelength of the illumination light, m is the number of pixels in a period, p is the pixel size of the spatial light modulator (5), and + or - depends on the slope direction of the linear phase.

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