Near-field tomography microscopic imaging method based on total internal reflection digital holography

Abstract

The invention belongs to the technical field of optical microscopic three-dimensional imaging, and particularly relates to a near-field tomography microscopic imaging method based on total internal reflection digital holography. Under the condition that total internal reflection is met, the incident angle of laser is changed from large to small, evanescent waves with the penetration depth from shallow to deep are excited to illuminate a sample, and the reflection phase shift difference distribution of reflected light waves when the sample exists or not is detected by using the secondary exposure digital holographic interferometry. And a Fresnel formula is combined to calculate a theoretical curve of the refractive index of a near-field region sample relative to the reflection phase shift difference under different penetration depths, and the refractive index distribution of the sample is demodulated pixel by pixel and layer by layer, so that wide-field and label-free near-field three-dimensional imaging is realized.

Description

Technical Field

[0001] The present invention relates to the field of optical microscopic three-dimensional imaging, in particular to the fields of near-field microscopic imaging and digital holographic microscopic imaging based on total internal reflection, and specifically relates to a near-field tomographic microscopic imaging method based on total internal reflection digital holography. Background Art

[0002] When light waves enter a rarer medium from a denser medium at an angle greater than the critical angle, total internal reflection (TIR) occurs at the interface between the two media, and an evanescent wave with an amplitude that rapidly decays perpendicular to the interface is generated. Total internal reflection microscopy uses the evanescent wave with a penetration depth of the order of the wavelength for illumination, enabling the detection of sample information in the near-field region and is widely used in fields such as biochemistry and cell biology. Digital holographic microscopy (DHM) combines digital holographic interferometry with optical microscopy and is a commonly used quantitative phase imaging method. Due to its advantages such as real-time, non-destructive, non-invasive, and full-field measurement, digital holographic microscopy is widely used in fields such as biomedicine, industrial inspection, and micro-nano measurement. In 2016, Zhang et al. proposed a transmission / total internal reflection integrated digital holographic microscopy (J. Zhang, et al. “Transmission and total internal reflection integrated digital holographic microscopy,” Optic Letters, 41(16), 3844 - 3847(2016)), which achieved the simultaneous dynamic measurement of the two-dimensional distribution of the refractive index and thickness of dielectric samples. However, this method is only applicable to samples with a longitudinally uniform refractive index distribution and cannot achieve tomography. To achieve three-dimensional imaging of samples in the near-field region, various tomography technical solutions have been proposed. For example, Bohannon et al. proposed to detect the curve of the change in intensity with the incident angle (K.P. Bohannon, et al. “Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy,” Microscopy and Microanalysis, 23(5), 978 - 988(2017)) to demodulate the spatial distribution patterns of the refractive index and thickness pixel by pixel. However, this method is based on intensity measurement, and scattering, interference fringes, and artifacts result in poor imaging effects.In addition, Boulanger et al. proposed multi-angle total internal reflection fluorescence microscopy (MA-TIRFM). By acquiring dozens of TIRFM images at different incident angles, the mapping relationship between the illumination light field, the sample distribution, and the acquired images is constructed, and the three-dimensional image of the sample is solved by means of an inverse problem optimization algorithm (J. Boulanger, et al. “Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging,” Proceedings of the National Academy of Sciences, 111(48), 17164-17169 (2014)). Although this method can achieve near-field tomography through reconstruction and has a relatively high axial resolution, due to the use of fluorescent labeling, it can ultimately only obtain the three-dimensional distribution of fluorescent molecules, with problems such as limited selection of fluorescent probes and photobleaching in long-term live cell imaging. Therefore, it is of great significance to develop a near-field tomography microscopy method. Summary of the Invention

[0003] Technical problems to be solved

[0004] To overcome the deficiencies of existing methods and technologies and achieve wide-field, label-free near-field three-dimensional imaging, the present invention proposes a near-field tomography microscopy method based on total internal reflection digital holography.

[0005] The idea of the present invention is as follows: First, under the condition of total internal reflection, the incident angle is changed from large to small to excite evanescent waves with different penetration depths from shallow to deep to illuminate the sample, and a series of image stacks with different illumination depths are obtained; Second, the reflection phase shift difference distribution of the reflected light wave with / without the sample is measured by using double-exposure digital holographic interferometry, and combined with the Fresnel formula, the theoretical curve of the refractive index of the sample in the near-field region relative to the reflection phase shift difference at different penetration depths is calculated. The refractive index corresponding to the closest theoretical value to the experimental value is used as the demodulation value, and the refractive index distribution is demodulated pixel by pixel and layer by layer, and finally the quantitative measurement of the three-dimensional distribution of the refractive index of the sample in the near-field region is realized.

[0006] Technical solution

[0007] In a first aspect, a near-field tomography microscopy method based on total internal reflection digital holography is provided, including the following steps:

[0008] Step 1: Construct a total internal reflection structure consisting of a glass substrate and a sample with a refractive index smaller than that of the glass substrate. The sample is surrounded by an aqueous medium. Calculate the critical angle θ for total internal reflection at the glass substrate-aqueous medium interface. c ;

[0009] Step 2: When the c Under the premise of , N angles are taken at each interval Δθ as the incident angle θ of the total internal reflection structure i (i=1, 2…N), calculate the penetration depth d of the evanescent wave when total internal reflection occurs at the glass substrate-aqueous medium interface at the corresponding incident angle zi , with d corresponding to θ1 z1 As the first layer thickness d1, θ i The corresponding thickness of the i-th layer d i d zi -d z(i-1) , we get N groups of incident angles θ i and layer thickness d i The corresponding data;

[0010] Step 3: Use parallel light with a specific polarization state to measure the i The light is incident obliquely on the glass substrate-sample interface and undergoes total internal reflection. The reflected light wave carrying the sample information acts as the object light wave, which interferes with the reference light wave that is not modulated by the sample and forms a hologram H. i (i=1,2…N), and the phase distribution of the object light wave is reconstructed using the light wave diffraction theory i (x, y), recorded as the experimental step; after removing the sample, repeat the experimental step and record the corresponding background hologram H 0i And reconstruct the phase distribution φ 0i (x, y); (x, y) is a two-dimensional space coordinate;

[0011] Step 4: Use the phase distribution data obtained in step 3 to calculate the phase difference distribution Δ of the reflected light wave Φ i ( x , y )= φ i ( x , y )- φ 0i ( x , y ), Δ Φ i ( x , y ) is input into the demodulation algorithm to demodulate the sample's refractive index three-dimensional distribution pixel by pixel and layer by layer. n ( x , y ,z ) to achieve near-field tomography microscopy.

[0012] Further, in step 2, the N is determined by the refractive index of the experimental system and the aqueous medium. Among them, the refractive indices of the glass substrate and the aqueous medium in the experimental system determine the minimum angle θ of laser incidence min , and the numerical aperture of the experimental system determines the maximum angle θ of laser incidence max , and the galvanometer accuracy for controlling the incident angle determines the interval Δθ of the incident angle. Then N = (θ max - θ min ) / Δθ.

[0013] Further, in step 4, the working process of the demodulation algorithm is as follows:

[0014] When the incident angle of the light beam is θ1, the evanescent wave generated by total internal reflection only penetrates the first-layer sample with a thickness of d1. At this time, the reflection phase shift φ1(x, y) is only related to the refractive indices of the first-layer sample and the glass substrate, and the background reflection phase shift φ 01 (x, y) is only related to the refractive indices of the aqueous medium and the glass substrate. Given the refractive indices of the glass substrate and the aqueous medium, by theoretically fitting the relationship between the refractive index of the first-layer sample and the phase difference of the corresponding reflected light wave, the refractive index corresponding to the case where the theoretical value is closest to the experimental value ΔΦ1(x, y) is used as the refractive index of the first-layer sample;

[0015] When the incident angle decreases from θ1 to θ2, the penetration depth of the evanescent wave increases by d2 compared to d1. At this time, the reflection phase shift φ2(x, y) is related to the thickness and refractive index of the first-layer sample, the refractive indices of the second-layer sample and the glass substrate, and the background reflection phase shift φ 02 (x, y) is still only related to the refractive indices of the aqueous medium and the glass substrate. Taking the refractive index of the demodulated first-layer sample as known, the refractive index of the second-layer sample is demodulated by theoretical fitting; repeating the above steps can demodulate the three-dimensional refractive index distribution of the sample pixel by pixel and layer by layer.

[0016] In a second aspect, a system for implementing the near-field tomography microscopy method based on total internal reflection digital holography in the first aspect is provided.

[0017] Beneficial effects

[0018] Compared with the prior art, the present application has at least the following beneficial effects:

[0019] The near-field tomography microscopy imaging method based on total internal reflection digital holography proposed by the present invention records the object field and background holograms at different incident angles within a specific range in sequence, reconstructs and calculates the reflection phase shift difference distribution of the corresponding reflected light waves with / without the sample based on the double-exposure method, combines with the Fresnel formula to calculate the theoretical curve of the refractive index of the sample in the near-field region at different penetration depths with respect to the reflection phase shift difference, and takes the refractive index corresponding to the closest theoretical value and experimental value as the demodulation value, thereby demodulating the three-dimensional refractive index distribution of the sample pixel by pixel and layer by layer, so as to achieve wide-field, label-free near-field three-dimensional imaging. Description of the Drawings

[0020] Figure 1 It is the optical path diagram of the angle-scanning near-field digital holographic tomography microscopy imaging system related to the present invention;

[0021] Figure 2 It is a schematic diagram of total internal reflection occurring when incident at an angle of θ1 on the glass substrate-aqueous medium interface;

[0022] Figure 3 It is a schematic diagram of total internal reflection occurring when incident at an angle of θ2 on the glass substrate-aqueous medium interface;

[0023] Figure 4 It is a graph showing the variation relationship between the evanescent wave penetration depth and the incident angle at the glass substrate-water interface under total internal reflection illumination;

[0024] Figure 5 It is a schematic diagram of the corresponding layer thickness during angle scanning;

[0025] Figure 6 It is the principle block diagram of the demodulation algorithm;

[0026] Reference Signs:

[0027] 1 - He-Ne laser, 2 - attenuator, 3 - objective lens, 4 - pinhole, 5 - convex lens 1, 6 - one-dimensional galvanometer, 7 - polarizer, 8 - half-wave plate, 9 - convex lens 2, 10 - beam splitter prism, 11 - high numerical aperture oil immersion microscope objective, 12 - convex lens 3, 13 - Wollaston prism, 14 - polarizer, 15 - image acquisition device;

[0028] d is the theoretically calculated layer thickness, ΔΦ is the reflection phase shift difference distribution obtained experimentally, θ is the scanning angle, n1 is the refractive index of the glass substrate, n2 is the refractive index of the aqueous medium above the glass substrate, i, j are sequence numbers, m is the number of incident angles, which also represents the number of layers, E is the difference between the theoretical value δ of the reflection phase shift difference at different refractive indices and the experimental value ΔΦ, cols is the column value where the minimum E is located, ε is the permittivity, and n is the refractive index. Detailed Embodiment

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] The present invention will now be further described in combination with embodiments and the accompanying drawings:

[0031] Embodiment 1

[0032] A near-field tomography microscopy imaging method based on total internal reflection digital holography, characterized by including the following steps:

[0033] Step 1: Construct a total internal reflection structure composed of a glass substrate and a sample with a refractive index less than that of the glass substrate, with an aqueous medium surrounding the sample, and calculate the critical angle θ of total internal reflection occurring at the glass substrate-aqueous medium interface according to Snell's law c ;

[0034] Step 2: On the premise of being greater than θ c , take N angles at intervals of Δθ as the incident angles θ i (i = 1, 2... N) of the total internal reflection structure respectively. For example, take angles from large to small at intervals of 0.1°, and calculate the penetration depth d of the evanescent wave when total internal reflection occurs at the glass substrate-aqueous medium interface at the corresponding incident angle zi . Take the d z1 corresponding to θ1 as the thickness d1 of the first layer, and the thickness d i of the i-th layer corresponding to θ i is d zi - d z(i-1) , to obtain N groups of corresponding data of the incident angle θ i and the stratified thickness d i ;

[0035] The N is determined by the experimental system and the refractive index of the aqueous medium. Among them, the refractive indices of the glass substrate and the aqueous medium in the experimental system determine the minimum angle θ min of laser incidence, the numerical aperture of the experimental system determines the maximum angle θ max of laser incidence, and the galvanometer accuracy for controlling the incident angle determines the interval Δθ of the incident angle. Then N = (θ max - θ min ) / Δθ. The larger N is, the more stratification numbers there are, and the more accurate the tomography measurement result is;

[0036] Step 3: Use parallel light with a specific polarization state to obliquely incident on the glass substrate-sample interface at θ i respectively and undergo total internal reflection. Among them, the reflected light wave carrying the sample information serves as the object light wave, and interferes with the reference light wave not modulated by the sample to form a hologram H i(i = 1, 2…N), the phase distribution φ of the object light wave is reconstructed using the light wave diffraction theory i (x, y), which is recorded as the experimental procedure; after removing the sample, repeat this experimental procedure and record the corresponding background hologram H 0i and reconstruct to obtain the phase distribution φ 0i (x, y); where (x, y) are two-dimensional spatial coordinates;

[0037] Step 4: Calculate the phase difference distribution of the reflected light wave using the phase distribution data obtained in Step 3:

[0038] ΔΦ i (x, y) = φ i (x, y) - φ 0i (x, y)

[0039] Input ΔΦ i (x, y) into the demodulation algorithm to demodulate the three-dimensional refractive index distribution n(x, y, z) of the sample pixel by pixel and layer by layer;

[0040] The working principle of the demodulation algorithm is as follows: When the incident angle of the light beam is θ1, the evanescent wave generated by total internal reflection only penetrates the first layer of the sample with a thickness of d1. At this time, the reflection phase shift φ1(x, y) is only related to the refractive indices of the first layer of the sample and the glass substrate, and the background reflection phase shift φ 01 (x, y) is only related to the refractive indices of the aqueous medium and the glass substrate. Since the refractive indices of the glass substrate and the aqueous medium are known, by theoretically fitting the relationship between the refractive index of the first layer of the sample and the corresponding phase difference of the reflected light wave, the refractive index corresponding to when the theoretical value is closest to the experimental value ΔΦ1(x, y) is used as the refractive index of the first layer of the sample; when the incident angle decreases from θ1 to θ2, the penetration depth of the evanescent wave increases by d2 compared to d1, that is, the thickness of the second layer described in Step 2. At this time, the reflection phase shift φ2(x, y) is related to the thickness and refractive index of the first layer of the sample, the refractive indices of the second layer of the sample and the glass substrate, and the background reflection phase shift φ 02 (x, y) is still only related to the refractive indices of the aqueous medium and the glass substrate. Taking the refractive index of the demodulated first layer of the sample as known, the refractive index of the second layer of the sample is demodulated by theoretical fitting; repeating the above steps can demodulate the three-dimensional refractive index distribution of the sample pixel by pixel and layer by layer, realizing near-field tomographic microscopy.

[0041] Example 2

[0042] This example provides a system for implementing the near-field tomographic microscopy method based on total internal reflection digital holography described in Implementation 1.

[0043] Figure 1Shown is an experimental system for angle-scanning near-field digital holographic tomography microscopy. The laser beam emitted by the He-Ne laser 1 (wavelength 632.8 nm) is expanded and collimated into a parallel light beam after passing through the attenuator 2, the objective lens 3, the pinhole 4, and the convex lens 5. This parallel light beam passes through the polarizer 7, the half-wave plate 8, the convex lens 9, and the beam splitter prism 10 and then converges to the rear focal plane of the oil-immersion objective lens 11. After being collimated by the objective lens, it is incident on the glass substrate-sample interface in a wide-field illumination manner and undergoes total internal reflection. The one-dimensional galvanometer 6 adjusts the incident angle of the incident light beam, and the polarizer modulates the polarization state of the incident light beam into 45° linearly polarized light, including s and p polarization components. The reflected light passes through the beam splitter prism 10 and the convex lens 12 (with the same focal length as the convex lens 9), and then off-axis interference is achieved through the Wollaston prism 13 and the polarizer 14. The formed hologram is received by the image acquisition device 15. Among them, the sample is moved to one side of the illumination area so that when total internal reflection is excited, half of the illumination area is the sample area and the other half is the background area. The reflected light is divided into two orthogonally polarized light beams after passing through the Wollaston prism 13. The light wave carrying the sample information serves as the object light wave, and the light wave not modulated by the sample serves as the reference light wave. After passing through the polarizer 14, the two light beams undergo off-axis interference in the overlapping area.

[0044] Before starting the experiment, calculate the critical angle θ of total internal reflection at the glass substrate-aqueous medium interface according to Snell's law. c , on the premise of being greater than θ c , take N angles from large to small at intervals of 0.1° as the incident angles θ of the total internal reflection structure i (i = 1, 2... N). Calculate the penetration depth d of the evanescent wave when total internal reflection occurs at the glass substrate-aqueous medium interface at the corresponding incident angle. zi , according to the principle of angle-scanning total internal reflection, θ c is the critical angle of total internal reflection at the glass substrate-aqueous medium interface. Figure 2 When incident on the glass substrate-aqueous medium interface at an angle of θ1 and total internal reflection occurs, the penetration depth of the evanescent wave is d z1 , which is used as the first layer thickness d1. Figure 3 When incident on the glass substrate-aqueous medium interface at an angle of θ2 and total internal reflection occurs, the penetration depth of the evanescent wave is d z2 , and the difference between d2 and d1 is used as the second layer thickness.

[0045] Take the d corresponding to θ1 z1 as the first layer thickness d1, and the thickness d of the i-th layer corresponding to θ i as d i as d zi - d z(i-1) , to obtain N sets of corresponding data of the incident angle θ i and the stratified thickness d i .

[0046] As Figures 4 - 5 shown, Figure 4 Figure 1 shows the relationship between the penetration depth of the evanescent wave at the glass substrate-water interface and the incident angle under total internal reflection illumination. Figure 5 Figure 2 is a schematic diagram of the corresponding layer thickness during angle scanning. Taking total internal reflection occurring at the glass substrate-water interface as an example, the penetration depth of the evanescent wave and the corresponding layer thickness during angle scanning are theoretically calculated respectively. The wavelength of the incident light λ is 632.8 nm, the refractive index of the glass is 1.5151, the refractive index of water is 1.3317, and the incident angle changes every 0.1° within the range of 62° to 70°.

[0047] In the experiment, parallel light with a specific polarization state is obliquely incident on the glass substrate-sample interface at an angle of θ i and total internal reflection occurs. Among them, the reflected light wave carrying the sample information serves as the object light wave, interferes with the reference light wave not modulated by the sample, and forms a hologram H i (i = 1, 2…N). The phase distribution φ i (x, y) of the object light wave is reconstructed using the light wave diffraction theory. After removing the sample, repeat the experimental steps, record the corresponding background hologram H 0i and reconstruct the phase distribution:

[0048] φ 0i (x, y). Then, the phase difference distribution ΔΦ i (x, y) of the reflected light wave is calculated using the obtained phase distribution data, and ΔΦ i (x, y)=φ 0i (x, y)-φ i (x, y). The three-dimensional refractive index distribution n(x, y, z) of the sample is demodulated pixel by pixel and layer by layer by inputting ΔΦ

[0049] As Figure 6 shown, when demodulating, the refractive index n1 of the glass substrate, the refractive index n2 of the aqueous medium, the theoretically calculated layer thickness d, the experimentally obtained reflection phase shift difference distribution ΔΦ, and the corresponding incident angle θ are used as calculation parameters. The search range of the refractive index is set to 1.2 to 1.9, and the accuracy is 0.0001. Within a single pixel, the refractive index is default to be evenly distributed along each longitudinal layer. When the incident angle of the light beam is θ1, the evanescent wave generated by total internal reflection only penetrates the first layer of the sample with a thickness of d1. At this time, the reflection phase shift φ1(x, y) is only related to the refractive indices of the first layer of the sample and the glass substrate, and the background reflection phase shift φ 01(x, y) is only related to the refractive indices of the aqueous medium and the glass substrate. Since the refractive indices of the glass substrate and the aqueous medium are known, by theoretically fitting the relationship between the refractive index of the first-layer sample and the corresponding phase difference of the reflected light wave, the refractive index corresponding to the case when the theoretical value is closest to the experimental value ΔΦ1(x, y) is taken as the refractive index of the first-layer sample; when the incident angle decreases from θ1 to θ2, the penetration depth of the evanescent wave increases by d2 compared to d1, that is, the thickness of the second layer. At this time, the reflection phase shift φ2(x, y) is related to the thickness and refractive index of the first-layer sample, the refractive indices of the second-layer sample and the glass substrate, and the background reflection phase shift φ 02 (x, y) is still only related to the refractive indices of the aqueous medium and the glass substrate. Taking the demodulated refractive index of the first-layer sample as known, the refractive index of the second-layer sample is demodulated by means of theoretical fitting; repeating the above steps can demodulate the three-dimensional refractive index distribution of the sample pixel by pixel and layer by layer, so as to realize wide-field, label-free near-field three-dimensional imaging.

[0050] The technical features of the above embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0051] The above embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. A near-field tomography microscopy imaging method based on total internal reflection digital holography, characterized in that Including the following steps: Step 1: Construct a total internal reflection structure composed of a glass substrate and a sample with a refractive index less than that of the glass substrate. The sample is surrounded by an aqueous medium, and calculate the critical angle θ for total internal reflection at the glass substrate-aqueous medium interface. c ; Step 2: When the c Under the premise of , N angles are taken at each interval Δθ as the incident angle θ of the total internal reflection structure i (i=1, 2…N), calculate the penetration depth d of the evanescent wave when total internal reflection occurs at the glass substrate-aqueous medium interface at the corresponding incident angle zi , with d corresponding to θ1 z1 As the first layer thickness d1, θ i The corresponding thickness of the i-th layer d i d zi -d z(i-1) , we get N groups of incident angles θ i and layer thickness d i The corresponding data; Step 3: Use polarized parallel light to obliquely incident on the glass substrate-sample interface at θ i respectively, and total internal reflection occurs. Among them, the reflected light wave carrying the sample information serves as the object light wave, interferes with the reference light wave not modulated by the sample, and forms a hologram H i (i = 1, 2…N), and the phase distribution φ of the object light wave is reconstructed using the light wave diffraction theory i (x, y), which is recorded as the experimental step; after removing the sample, repeat this experimental step, and record the corresponding background hologram H 0i and reconstruct the phase distribution φ 0i (x, y); (x, y) is the two-dimensional space coordinate; Step 4: Calculate the phase difference distribution ΔΦ of the reflected light wave from the phase distribution data obtained in Step 3 i (x,y)=φ i (x,y)-φ 0i (x,y), and input ΔΦ i (x,y) into the demodulation algorithm to demodulate the three-dimensional refractive index distribution n(x,y,z) of the sample pixel by pixel and layer by layer, so as to realize near-field tomography microscopy imaging.

2. The near-field tomography microscopy imaging method based on total internal reflection digital holography according to claim 1, characterized in that: In the said step 2, the value of N is determined by the experimental system and the refractive index of the aqueous medium. Among them, the refractive indices of the glass substrate and the aqueous medium in the experimental system determine the minimum angle θ of laser incidence min , and the numerical aperture of the experimental system determines the maximum angle θ of laser incidence max . The interval Δθ of the incident angle is determined by the galvanometer accuracy for controlling the incident angle. Then N = (θ max - θ min ) / Δθ.

3. A near-field tomography microscopy imaging method based on total internal reflection digital holography according to claim 1, characterized in that: The working process of the demodulation algorithm is as follows: When the incident angle of the light beam is θ1, the evanescent wave generated by total internal reflection only penetrates the first-layer sample with a thickness of d1. At this time, the reflection phase shift φ1(x, y) is only related to the refractive indices of the first-layer sample and the glass substrate, and the background reflection phase shift φ 01 (x, y) is only related to the refractive indices of the aqueous medium and the glass substrate. Given the refractive indices of the glass substrate and the aqueous medium, by theoretically fitting the relationship between the refractive index of the first-layer sample and the corresponding phase difference of the reflected light wave, the refractive index corresponding to the closest theoretical value and the experimental value ΔΦ1(x, y) is taken as the refractive index of the first-layer sample; When the incident angle decreases from θ1 to θ2, the penetration depth of the evanescent wave increases by d2 compared to d1. At this time, the reflection phase shift φ2(x,y) is related to the thickness and refractive index of the first-layer sample, and the refractive indices of the second-layer sample and the glass substrate. The background reflection phase shift φ 02 (x,y) is still only related to the refractive indices of the aqueous medium and the glass substrate. Taking the refractive index of the first-layer sample demodulated as known, the refractive index of the second-layer sample is demodulated by means of theoretical fitting; repeating the above steps can demodulate the three-dimensional refractive index distribution of the sample pixel by pixel and layer by layer.

4. A system for implementing the near-field tomography microscopy method based on total internal reflection digital holography according to any one of claims 1-3.

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