A near-field tomographic microscopy method based on total internal reflection digital holography

Abstract

The present invention belongs to the technical field of optical microscopic three-dimensional imaging, and specifically relates to a near-field tomographic microscopic imaging method based on total internal reflection digital holography. Under the condition of total internal reflection, the laser incident angle is varied from large to small, stimulating evanescent waves with penetration depths from shallow to deep to illuminate the sample. Double-exposure digital holographic interferometry is used to detect the reflection phase shift difference distribution of the reflected light wave with and without the sample. The Fresnel formula is combined to calculate theoretical curves of the sample refractive index in the near-field region relative to the reflection phase shift difference at different penetration depths. The sample refractive index distribution is then demodulated pixel by pixel and layer by layer, thereby achieving wide-field, label-free near-field three-dimensional imaging.

Description

Technical Field

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

[0002] When light waves enter an optically denser medium from an optically rarer medium at an angle greater than the critical angle, total internal reflection (TIR) ​​occurs at the interface between the two media, generating an evanescent wave whose amplitude decays sharply perpendicular to the interface. Total internal reflection microscopy utilizes evanescent waves with a wavelength-scale penetration depth 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), a commonly used quantitative phase imaging method that combines digital holographic interferometry with optical microscopy, is widely used in biomedicine, industrial testing, and micro-nanometry due to its real-time, non-destructive, non-invasive, and full-field measurement capabilities. 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 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 uniform refractive index distribution along the longitudinal direction and cannot achieve tomographic imaging. In order to achieve three-dimensional imaging of samples in the near-field region, various tomographic technology solutions have been proposed. For example, Bohannon et al. proposed to demodulate the spatial distribution pattern of refractive index and thickness pixel by pixel by detecting the curve of change of intensity and incident angle (KP 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)). However, this method is based on intensity measurement, where scattering, interference fringes, and artifacts lead to poor imaging results.In addition, Boulanger et al. proposed multi-angle total internal reflection fluorescence microscopy (MA-TIRFM), which constructs the mapping relationship between the illumination light field, sample distribution and collected image by acquiring dozens of TIRFM images at different incident angles, and solves the three-dimensional image of the sample with the help of 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 high axial resolution, it can only obtain the three-dimensional distribution of fluorescent molecules due to the use of fluorescent labels. There are 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] In order to overcome the shortcomings of existing methods and technologies and realize wide-field, label-free near-field three-dimensional imaging, the present invention proposes a near-field tomographic microscopy imaging method based on total internal reflection digital holography.

[0005] The idea of ​​the present invention is: first, under the condition of total internal reflection, the incident angle is changed from large to small, and evanescent waves with penetration depths from shallow to deep are stimulated to illuminate the sample, thereby obtaining a series of image stacks with different illumination depths; second, the reflection phase shift difference distribution of the reflected light wave with and without the sample is measured using double-exposure digital holographic interferometry, and the theoretical curve of the refractive index of the sample in the near-field area relative to the reflection phase shift difference at different penetration depths is calculated in combination with the Fresnel formula. 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, ultimately achieving quantitative measurement of the three-dimensional distribution of the refractive index of the sample in the near-field area.

[0006] Technical Solution

[0007] In a first aspect, a near-field tomographic microscopy method based on total internal reflection digital holography is provided, comprising 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 taking N angles at every 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 φ 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 tomographic microscopy.

[0012] Furthermore, in step 2, N is determined by the refractive index of the experimental system and the aqueous medium, wherein the refractive index of the glass substrate and the aqueous medium in the experimental system determines the minimum angle θ of the laser incident. min , the numerical aperture of the experimental system determines the maximum angle of laser incidence θ max , the accuracy of the galvanometer that controls the incident angle determines the interval Δθ of the incident angle, so N=(θ max -θ min ) / Δθ.

[0013] Furthermore, in step 4, the working process of the demodulation algorithm is:

[0014] 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 index of the first layer of the sample and the glass substrate. The background reflection phase shift φ 01 (x, y) is only related to the refractive index of the aqueous medium and the glass substrate. Given the refractive indices of the glass substrate and the aqueous medium, the relationship between the refractive index of the first layer of the sample and the corresponding phase difference of the reflected light wave is theoretically fitted. The refractive index corresponding to the closest theoretical value to the experimental value ΔΦ1(x, y) is taken as the refractive index of the first layer of the 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 of sample, the refractive index of the second layer of sample and the glass substrate. The background reflection phase shift φ 02 (x, y) is still only related to the refractive index of the aqueous medium and the glass substrate. The refractive index of the demodulated first layer of the sample is taken as known, and 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.

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

[0017] Beneficial effects

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

[0019] The near-field tomographic microscopy method based on total internal reflection digital holography proposed in the present invention sequentially records the object field and background holograms at different incident angles within a specific range, reconstructs and calculates the corresponding reflection phase shift difference distribution of the reflected light wave with and without a sample based on the double exposure method, and combines the Fresnel formula to calculate the theoretical curve of the sample refractive index in the near-field area relative to the reflection phase shift difference at different penetration depths. The refractive index corresponding to the closest theoretical value to the experimental value is used as the demodulation value, and the three-dimensional refractive index distribution of the sample is demodulated pixel by pixel and layer by layer, thereby realizing wide-field, label-free near-field three-dimensional imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an optical path diagram of the angle scanning near-field digital holographic tomography microscopy imaging system involved in the present invention;

[0021] Figure 2 Schematic diagram of total internal reflection at the glass substrate-aqueous medium interface at an angle of θ1;

[0022] Figure 3 Schematic diagram of total internal reflection at the glass substrate-aqueous medium interface at an angle of θ2;

[0023] Figure 4 This is a graph showing 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;

[0024] Figure 5 Schematic diagram of layer thickness corresponding to angle scanning;

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

[0026] Reference numerals:

[0027] 1-He-Ne laser, 2-attenuation plate, 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 experimentally obtained reflection phase shift difference distribution, 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 and j are the 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 and the experimental value ΔΦ of the reflection phase shift difference under different refractive indices, cols is the column value with the minimum E, ε is the dielectric constant, and n is the refractive index. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, 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 intended to limit the present invention.

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

[0031] Example 1

[0032] A near-field tomographic microscopy imaging method based on total internal reflection digital holography is characterized by comprising the following steps:

[0033] 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. According to Snell's law, calculate the critical angle θ of total internal reflection at the glass substrate-aqueous medium interface. c ;

[0034] Step 2: When the c Under the premise of taking N angles at every interval Δθ as the incident angle θ of the total internal reflection structure i (i=1,2…N), if the angle is taken from large to small at intervals of 0.1°, 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;

[0035] The N is determined by the refractive index of the experimental system and the aqueous medium. The refractive index of the glass substrate and the aqueous medium in the experimental system determines the minimum angle θ of the laser incident. min , the numerical aperture of the experimental system determines the maximum angle of laser incidence θ max , the accuracy of the galvanometer that controls the incident angle determines the interval Δθ of the incident angle, so N=(θ max -θ min ) / Δθ, the larger N is, the more layers there are, and the more accurate the tomographic measurement results are;

[0036] 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 φ 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;

[0037] Step 4: Use the phase distribution data obtained in step 3 to calculate the phase difference distribution of the reflected light wave:

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

[0039] ΔΦ i (x, y) is input into the demodulation algorithm to demodulate the sample's three-dimensional refractive index distribution n(x, y, z) 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 index 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 index of the aqueous medium and the glass substrate. Since the refractive indices of the glass substrate and the aqueous medium are known, the relationship between the refractive index of the first layer of the sample and the corresponding phase difference of the reflected light wave is theoretically fitted, and the refractive index corresponding to the closest theoretical value to the experimental value ΔΦ1(x, y) is taken 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 index 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 index of the aqueous medium and the glass substrate. The refractive index of the demodulated first layer of the sample is taken as known, and 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 imaging.

[0041] Example 2

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

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

[0044] Before starting the experiment, the critical angle θ at which total internal reflection occurs at the interface between the glass substrate and the aqueous medium is calculated according to Snell's law. c , when greater than θ c Under the premise of , N angles are taken from large to small at intervals of 0.1° 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 , 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 the evanescent wave is incident at an angle of θ1 and undergoes total internal reflection at the glass substrate-aqueous medium interface, the penetration depth is d z1 , as the first layer thickness d1, Figure 3 When the evanescent wave is incident at an angle of θ2 and undergoes total internal reflection at the glass substrate-aqueous medium interface, the penetration depth is d z2 , take the difference between d2 and d1 as the thickness of the second layer.

[0045] The d corresponding to θ1 z1 As the first layer thickness d1, θ i The corresponding thickness of the i-th layer d i As d zi -d z(i-1) , we get N groups of incident angles θ i and layer thickness d i The corresponding data.

[0046] like Figure 4-5 As shown, Figure 4 This is 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 3 is a schematic diagram of the corresponding layer thickness during angle scanning. Taking total internal reflection at the glass substrate-water interface as an example, the evanescent wave penetration depth and the corresponding layer thickness during angle scanning are theoretically calculated. The incident light wavelength λ is 632.8nm, the refractive index of glass is 1.5151, the refractive index of water is 1.3317, and the incident angle varies in intervals of 0.1° within the range of 62° to 70°.

[0047] The experiment uses parallel light with 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 φ of the object light wave is reconstructed using the light wave diffraction theory i (x, y), remove the sample and repeat the experimental steps to record the corresponding background hologram H 0i And reconstruct the phase distribution:

[0048] φ 0i (x, y), and then use the obtained phase distribution data to calculate the reflected light wave phase difference distribution ΔΦ i (x,y)=φ i (x,y)-φ 0i (x,y), ΔΦ i (x, y) is input into the demodulation algorithm and the three-dimensional refractive index distribution n(x, y, z) of the sample is demodulated pixel by pixel and layer by layer.

[0049] like Figure 6 As shown in the figure, during demodulation, 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 1.2~1.9, and the accuracy is 0.0001. Within a single pixel, the default refractive index is uniformly distributed along each layer in the longitudinal direction. 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 index 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 index of the aqueous medium and the glass substrate. Since the refractive indices of the glass substrate and the aqueous medium are known, the relationship between the refractive index of the first layer of the sample and the corresponding phase difference of the reflected light wave is theoretically fitted, and the refractive index corresponding to the closest theoretical value to the experimental value ΔΦ1(x, y) is taken 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. 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 index 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 index of the aqueous medium and the glass substrate. The refractive index of the demodulated first layer of the sample is taken as known, and 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, thereby realizing wide-field, label-free near-field three-dimensional imaging.

[0050] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0051] The above embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A near-field tomographic microscopy method based on total internal reflection digital holography, characterized in that The steps include: 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 ; Step 2: When the c Under the premise of taking N angles at every 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 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; Step 4: Use the phase distribution data obtained in step 3 to calculate the phase difference distribution ΔΦ of the reflected light wave i (x,y)=φ i (x,y)-φ 0i (x,y), ΔΦ i (x, y) is input into the demodulation algorithm to demodulate the sample's three-dimensional refractive index distribution n(x, y, z) pixel by pixel and layer by layer to achieve near-field tomographic microscopy imaging. 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 of the sample with a thickness of d1. At this time, the reflection phase shift φ1(x, y) is only related to the refractive index of the first layer of the sample and the glass substrate. The background reflection phase shift φ 01 (x, y) is only related to the refractive index of the aqueous medium and the glass substrate. Given the refractive indices of the glass substrate and the aqueous medium, the relationship between the refractive index of the first layer of the sample and the corresponding phase difference of the reflected light wave is theoretically fitted. The refractive index corresponding to the closest theoretical value to the experimental value ΔΦ1(x, y) is taken 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. At this time, the reflection phase shift φ2(x, y) is related to the thickness and refractive index of the first layer of sample, the refractive index of the second layer of sample and the glass substrate. The background reflection phase shift φ 02 (x, y) is still only related to the refractive index of the aqueous medium and the glass substrate. The refractive index of the demodulated first layer of the sample is taken as known, and 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.

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

3. A system for implementing the near-field tomographic microscopy method based on total internal reflection digital holography according to any one of claims 1 to 2.

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