An optical tweezers longitudinal positioning feedback device and method based on fluorescence coupling emission

Abstract

The present invention discloses an optical tweezer longitudinal positioning feedback device and method based on fluorescence coupling emission. The device includes: a spectroscope, an objective lens, an imaging lens, an imaging sensor, an immersion solution, a fluorescent sample, a multi-layer slide, a collecting lens, a signal acquisition sensor, a front lens and a displacement control device. The method is as follows: a multi-layer slide with a mode coupling emission function is prepared; the laser used to capture the sample is expanded and then reflected and focused to the fluorescent sample in the immersion solution by the spectroscope and the objective lens; the light emitted by the fluorescent sample is emitted downward at different angles through the multi-layer slide; after being focused on the signal acquisition sensor by the collecting lens, the ratio of the emission light intensity between different angle regions is calculated; the longitudinal position of the fluorescent sample is calculated according to the ratio and a position criterion is provided for the device, and then the position of the front lens is controlled by the displacement control device. The present invention solves the technical problem of low longitudinal positioning accuracy of fluorescent samples in traditional optical tweezers systems.

Description

Technical Field

[0001] The invention relates to the technical field of imaging and optical tweezers operation, and in particular to an optical tweezers longitudinal positioning feedback device and method based on fluorescence coupled emission. Background Art

[0002] Optical tweezers are generally a technology that uses the light pressure in a tightly focused laser to form scattering force and gradient force, and to capture and manipulate tiny particles and cells. Its non-contact and non-destructive characteristics have great application potential in biomedicine and microscopic imaging, and have greatly promoted the development of multidisciplinary and interdisciplinary fields. As a far-field method, it can design, manipulate and fine-tune the light beam with nanometer precision through advanced processing methods and precise mechanical control, but the positioning of the captured nanoparticles is still inaccurate. It can only provide an effective control method but it is difficult to reflect the specific position of the nanoparticles after the light beam acts on the particles. Moreover, due to the complexity and diversity of the samples, the designed light beam will also produce certain errors after various scatterings. Because of this, the optical tweezers technology used in photophysics and biomedicine often cannot get timely feedback on the capture effect, making it difficult for micromanipulation technology to be more widely used in actual use. Conventional means to solve such problems often require more time and the use of complex equipment to improve imaging resolution, and use super-resolution imaging technology to further locate the sample. However, how to efficiently and quickly locate the sample and dynamically adjust the light beam has always been a problem to be solved.

[0003] For the dynamic control of the light beam in optical tweezers, most of the general implementation methods are to use a highly sensitive positioning platform and match it with an existing commercial microscope. Based on the principle of optical tweezers, the force exerted by the focused laser beam on the particle is divided into two parts. The axial optical force along the propagation direction of the beam generally controls the nanoparticles near the substrate; while the radial optical force perpendicular to the axis of the beam generally has a Gaussian distribution of intensity and is an attractive force, which guides the tiny particles to the optical axis and constrains them near the center of the optical axis. That is, the radial component of the optical force can effectively confine the tiny particles in a two-dimensional area, so by adjusting the intensity of the laser beam, the position of the tiny particles in the two-dimensional area can be well controlled. Even if its half-maximum full width cannot break through the diffraction limit, its modulation depth is proportional to the total power of the beam, so by adjusting the beam power, the required lateral positioning accuracy can be improved, which can theoretically be reduced to a few nanometers. However, it can only be controlled and adjusted within the plane, and cannot achieve high-resolution information feedback in the longitudinal direction. At the same time, the required platform facilities are expensive and difficult to expand, which limits the sample testing in the laboratory stage. Nowadays, computer image analysis and recognition technology is used to track and restore blurred samples. It can only be optimized based on existing imaging, and cannot truly and completely and effectively achieve effective positioning of the longitudinal position. Therefore, conventional methods currently have certain limitations, which is not conducive to those skilled in the art to better apply optical tweezers technology to biological, medical and experimental applications. Summary of the invention

[0004] The present invention provides an optical tweezers longitudinal positioning feedback device and method based on fluorescence coupling emission to solve the problem of low positioning accuracy of the longitudinal position of a sample when dynamically adjusting a light beam in traditional optical tweezers technology.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] An optical tweezers longitudinal positioning feedback device based on fluorescence coupled emission comprises a beam splitter, an objective lens, an imaging lens, an imaging sensor, an immersion solution, a multi-layer slide, a collecting lens, a signal acquisition sensor, a front lens and a displacement control device. The imaging sensor, imaging lens, beam splitter, objective lens, multi-layer slide, collecting lens and signal acquisition sensor are arranged in sequence from top to bottom along the optical axis. The immersion solution is located above the multi-layer slide, and the fluorescent sample is suspended in the immersion solution. The front lens is arranged on one side of the beam splitter, and the front lens is connected to the displacement control device. The displacement control device can adjust the position of the front lens. The laser used to capture the fluorescent sample is expanded by the front lens, reflected by the beam splitter, and then focused by the objective lens above the multi-layer slide to capture the fluorescent sample in the immersion solution. The light emitted by the fluorescent sample is emitted downward at different angles through the multi-layer slide.

[0007] Furthermore, the displacement control device is connected to the acquisition sensor, and the displacement control device changes the position of the front lens according to the signal received by the signal acquisition sensor.

[0008] Furthermore, the multi-layer slide comprises a polymer layer, a metal layer and a dielectric layer arranged from top to bottom.

[0009] Furthermore, the polymer layer is a polymethyl methacrylate layer, and its thickness is 5nm to 10nm.

[0010] Furthermore, the dielectric layer has a nanometer-level thickness and is composed of dielectric materials with different refractive indices.

[0011] Furthermore, a high-pass filter is provided in the signal acquisition sensor, and the high-pass filter is used to filter out laser.

[0012] Furthermore, a narrow-band filter is provided at the entrance of the signal acquisition sensor to ensure that the collected signal is monochromatic fluorescence.

[0013] The longitudinal positioning feedback method using the optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission described above comprises the following steps:

[0014] Step 1: Prepare a corresponding multilayer slide according to the selected fluorescent sample, and drop an immersion solution containing the fluorescent sample on the prepared multilayer slide;

[0015] Step 2: Observe the sample and the laser in the imaging sensor, adjust the beam splitter and the objective lens, the laser used to capture the fluorescent sample is expanded by the front lens, reflected by the beam splitter, and then focused by the objective lens to the top of the multilayer slide and capture the fluorescent sample in the immersion solution, and record the position of the laser focus point. The light emitted by the fluorescent sample is emitted downward at different angles through the multilayer slide. Due to the modulation of the multilayer slide, the radiated fluorescence will be mainly concentrated in several different angle areas, and the angle corresponding to the weakest point of fluorescence radiation between different angle areas is taken as the demarcation angle;

[0016] Step three: Collect the fluorescence inside and outside the dividing angle through the collecting lens and the signal acquisition sensor, measure the ratio of the fluorescence intensity collected in different angle areas, calculate the fluorescence radiation intensity of the dipoles at different depths in each angle area according to the structure of the prepared multilayer slide and the dipole radiation theory, and obtain the relationship between the radiation fluorescence intensity ratio and the longitudinal depth. Then, according to the measured fluorescence intensity ratio, the longitudinal position of the fluorescent sample in the immersion solution is obtained, and the signal is fed back to the displacement control device, thereby changing the position of the front lens, and repeating this process to complete the precise positioning of the longitudinal position.

[0017] Furthermore, the preparation method of the multilayer slide in step 1 is: alternately depositing dielectric layers with different refractive indices on a transparent substrate, then evaporating a metal layer, then spin coating a PMMA polymer layer, and finally drying.

[0018] Furthermore, the demarcation angle in step three is selected at different angles according to the structure of the multi-layer slide.

[0019] In the traditional optical tweezers technology, the present invention replaces the glass slide with a multi-layer slide and adds an additional fluorescence collection device underneath, so that the system can collect the fluorescence coupled emission signal while the optical tweezers are capturing, and then measure the intensity ratio of different coupled emission modes. Combined with the designed multi-layer slide structure and the change of the light beam during the fine-tuning process, the longitudinal position of the fluorescent sample is accurately calculated and fed back to the electric control precision displacement stage, thereby realizing accurate dynamic feedback in the optical tweezers system.

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] (1) High-precision longitudinal positioning: The introduction of multilayer slides into the traditional optical tweezers technology and the expansion of the optical tweezers system provide a new calibration feedback method. While observing and capturing the fluorescent sample, the coupled emission intensity can be measured by the collection device. Combined with the structural characteristics of the multilayer slide, high-precision longitudinal positioning can be achieved at any absolute position in the immersion solution.

[0022] (2) Simple structure and low cost: No complex confocal components in commercial microscope systems are required, and no special fluorescent materials are required for super-resolution imaging. Only the inherent multi-layer structure of the multi-layer slide and a simple collection device can be used to observe micro-longitudinal displacements. At the same time, the signal is fed back to the front lens to adjust the light beam in time. At the same time, adding a front lens in front of the objective lens is also conducive to the efficient focusing of various complex vector beams when captured by optical tweezers.

[0023] (3) Easy to expand and has a wide range of applications: The optical tweezers longitudinal positioning feedback device based on fluorescence coupled emission in the present invention can be easily attached to the existing optical tweezers device, and can realize real-time observation of the longitudinal position of various fluorescent samples in traditional light binding and light capture. In the optical tweezers system, the light beam position can be conveniently and dynamically adjusted to improve the existing sensing accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic structural diagram of the optical tweezers longitudinal positioning feedback device based on fluorescence coupled emission of the present invention.

[0025] Figure 2 FIG. 4 is a relationship diagram between the longitudinal position and the ratio of the outer to inner boundary angle calculated according to the multi-layer slide structure in the embodiment.

[0026] Figure 3 This is a flow chart of step three of the optical tweezers longitudinal positioning feedback method based on fluorescence coupled emission of the present invention.

[0027] The reference numerals in the figure are: 1. spectroscope, 2. objective lens, 3. imaging lens, 4. imaging sensor, 5. immersion solution, 6. fluorescent sample, 7. multilayer slide, 8. collecting lens, 9. signal acquisition sensor, 10. front lens, 11. displacement control device. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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.

[0029] like Figure 1 As shown, an optical tweezers longitudinal positioning feedback device based on fluorescence coupled emission includes a spectroscope 1, an objective lens 2, an imaging lens 3, an imaging sensor 4, an immersion solution 5, a multilayer slide 7, a collecting lens 8, a signal acquisition sensor 9, a front lens 10 and a displacement control device 11. The imaging sensor 4, the imaging lens 3, the spectroscope 1, the objective lens 2, the multilayer slide 7, the collecting lens 8 and the signal acquisition sensor 9 are arranged in sequence from top to bottom along the optical axis, the immersion solution 5 is placed above the multilayer slide 7, the fluorescent sample 6 is suspended in the immersion solution 5, the front lens 10 is arranged on one side of the spectroscope 1, and the front lens 10 is connected to the displacement control device 11, and the displacement control device can control the position of the front lens 10. Among them, the multi-layer slide 7 is preferably a multi-layer glass slide, and the displacement control device 11 is preferably an electrically controlled precision displacement stage. The laser used to capture the fluorescent sample 6 is expanded by the front lens 10, reflected by the beam splitter 1, and then focused by the objective lens 2 to the top of the multi-layer slide 7 and capture the fluorescent sample 6 in the immersion solution. The light emitted by the fluorescent sample 6 passes through the multi-layer slide 7 and is emitted downward at different angles.

[0030] The displacement control device 11 is connected to the acquisition sensor 9 , and the displacement control device 11 changes the position of the front lens 10 according to the signal received by the signal acquisition sensor 9 .

[0031] The multilayer slide 7 includes a polymer layer, a metal layer and a dielectric layer arranged from top to bottom. The polymer layer is preferably a polymethyl methacrylate layer with a thickness of 5nm to 10nm; the metal layer has a thickness of 40nm to 50nm. The dielectric layer has a nanometer-level thickness and is composed of dielectric materials with different refractive indices. This ensures that part of the fluorescence emitted by the fluorescent sample is emitted within the demarcation angle, and part is emitted outside the demarcation angle.

[0032] The fluorescent sample can be located at any position in the immersion solution. When the fluorescent sample is located at different longitudinal positions in the immersion solution, the intensity ratio of the fluorescence collected by the signal acquisition sensor within the demarcation angle and outside the demarcation angle is different.

[0033] The signal acquisition sensor 9 is provided with a high-pass filter for filtering out laser light, thereby ensuring that the collected optical signals are pure fluorescent signals.

[0034] A narrow-band filter is provided at the entrance of the signal acquisition sensor 9 to ensure that the collected signal is monochromatic fluorescence.

[0035] The numerical aperture of the collecting lens is large enough to collect the fluorescence inside and outside the demarcation angle. Alternatively, the collecting lens can be replaced by an objective lens to collect the fluorescence inside and outside the demarcation angle.

[0036] The longitudinal positioning feedback method using the optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission specifically comprises the following steps:

[0037] Step 1: Prepare a corresponding multilayer slide 7 according to the selected fluorescent sample 6, and drop the immersion solution 5 containing the fluorescent sample 6 onto the prepared multilayer slide 7;

[0038] Step 2: Observe the sample and the laser in the imaging sensor 4, adjust the beam splitter 1 and the objective lens 2, the laser used to capture the fluorescent sample is expanded by the front lens 10, reflected by the beam splitter 1, and then focused by the objective lens 2 to the top of the multilayer slide 7 and capture the fluorescent sample 6 in the immersion solution, and record the position of the laser focus point. The light emitted by the fluorescent sample 6 is emitted downward at different angles through the multilayer slide 7. Due to the modulation of the multilayer slide 7, the radiated fluorescence will be mainly concentrated in several different angle areas, and the angle corresponding to the weakest point of fluorescence radiation between different angle areas is taken as the demarcation angle;

[0039] Step three: collect the fluorescence inside and outside the dividing angle through the collecting lens 8 and the signal collection sensor 9, measure the ratio of the fluorescence intensity collected in different angle areas, calculate the fluorescence radiation intensity of the dipoles at different depths in each angle area according to the structure of the prepared multilayer slide 7 and the dipole radiation theory, and obtain the relationship between the radiation fluorescence intensity ratio and the longitudinal depth. Then, according to the measured fluorescence intensity ratio, the longitudinal position of the fluorescent sample 6 in the immersion solution 5 is obtained, and the signal is fed back to the displacement control device 11, thereby changing the position of the front lens 10, and repeating this process to complete the precise positioning of the longitudinal position.

[0040] The method for preparing the multilayer slide 7 in step 1 comprises: alternately depositing dielectric layers with different refractive indices on a transparent substrate, then evaporating a metal layer, then spin coating a polymer layer, and finally drying.

[0041] The demarcation angle in step 3 is selected at different angles according to the different structures of the multilayer slide 7 .

[0042] The precise positioning of the longitudinal position of the fluorescent sample in the immersion solution in the above step 3 needs to be determined in combination with the laser focal plane observed during the coarse adjustment and the measured fluorescence intensity ratio. The approximate position range of the fluorescent sample is determined according to the focal plane position, and then the front lens is fine-tuned by the electronically controlled precision displacement stage to fine-tune the focus of the light beam up and down. At the same time, the trend of the fluorescence intensity ratio change is used to determine the movement direction of the fluorescent sample and its precise position. Thus, the signal is fed back to the electronically controlled precision displacement stage again to complete the precise dynamic control of the fluorescent sample in the optical tweezers experiment.

[0043] Example 1

[0044] An optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission comprises a beam splitter 1, an objective lens 2, an imaging lens 3, an imaging sensor 4, an immersion solution 5, a fluorescent sample 6, a multi-layer glass slide 7, a collecting lens 8, a signal acquisition sensor 9, a front lens 10 and an electrically controlled precision displacement stage 11. The imaging sensor 4, the imaging lens 3, the beam splitter 1, the objective lens 2, the multi-layer glass slide, the collecting lens 8 and the signal acquisition sensor 9 are sequentially arranged along the optical axis, the immersion solution 5 is placed above the multi-layer glass slide 7, the fluorescent sample 6 is suspended in the immersion solution 5, and the electrically controlled precision displacement stage 11 is connected to the front lens 10 and controls the position of the front lens 10.

[0045] Furthermore, the multi-layer slide glass 7 includes a polymer layer, a metal layer, and a dielectric layer arranged from top to bottom. The thickness of the dielectric layer of the multi-layer slide glass 7 can be changed according to the different fluorescent samples 6 to be tested.

[0046] Furthermore, the polymer layer of the multi-layer slide glass 7 is a polymethyl methacrylate layer, and the thickness of the layer is 10 nm.

[0047] Furthermore, the metal layer material of the multi-layer slide glass 7 is silver, and the thickness is 40 nm.

[0048] Furthermore, the dielectric layers of the multi-layer slide glass 7 are alternating silicon nitride layers and silicon dioxide layers, and the thickness of each layer does not exceed 200 nm.

[0049] Furthermore, the front lens 10 can change position according to the signal received by the electric control precision displacement stage. It is set that the high level signal moves to the right (away from the beam splitter 1) and the low level signal moves to the left (close to the beam splitter 1).

[0050] Further, the immersion solution 5 is an aqueous solution, and the fluorescent sample 6 is suspended in the immersion solution 5. When the fluorescent sample 6 is located at different longitudinal positions in the immersion solution 5, the ratio of the fluorescence collected by the signal acquisition sensor 9 is different inside and outside the demarcation angle.

[0051] Furthermore, the signal acquisition sensor 9 contains a high-pass filter to filter out the collected laser light. Additionally, a narrow-band filter is added before the signal acquisition sensor 9 to ensure that the collected signal is monochromatic fluorescence.

[0052] Furthermore, the collecting lens 8 is selected as an objective lens, which can collect the fluorescence inside and outside the boundary angle.

[0053] The longitudinal positioning feedback method of the optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission is adopted. In this embodiment, the sample is positioned 200 nm above the multi-layer slide. The following steps are included:

[0054] (1) preparing a multilayer glass slide according to the selected fluorescent sample 6 being a fluorescent ball doped with rhodamine 6G, the preparation of the multilayer glass slide comprising: alternately depositing silicon nitride and silicon dioxide on a transparent cover glass, then evaporating a metal layer, and finally spin coating a PMMA polymer layer, and then dropping an immersion solution 5 containing the fluorescent sample 6 on the top of the multilayer glass slide after drying;

[0055] (2) observing in the imaging sensor 4, adjusting the beam splitter 1, focusing the laser beam near the top of the multilayer slide and capturing the fluorescent sample 6, and determining the laser focal plane position, which is located near 200 nm above the multilayer slide;

[0056] (3) The fluorescence inside and outside the demarcation angle is collected by the collecting lens 8 and the signal collection sensor 9. The demarcation angle is taken as 58°. The initial ratio of the fluorescence intensity collected inside and outside the demarcation angle is measured to be 12.5. According to the structure of the multilayer slide glass 7 prepared in step 1, the fluorescence intensity ratio inside and outside the demarcation angle when the fluorescent sample is located in the immersion solution at a distance of 200 nm from the multilayer slide glass is calculated to be 11.93, which is defined as the reference value, such as Figure 2 First, the electronically controlled precision stage obtains a high-level signal, and then feeds back the initial value signal to the electronically controlled precision stage, thereby adjusting the position of the front lens 10 to the right and changing the position of the focused beam. The collected fluorescence intensity ratio is recorded and calculated again, and the position of the front lens is automatically fed back and adjusted again until the difference between the measured intensity ratio and the reference value is less than 0.01. This completes the precise positioning of the longitudinal position, such as Figure 3 .

[0057] This method uses the ratio of different emission intensities at different angles in the recorded sensor to accurately locate the position of the captured sample, with a theoretical error of less than 16nm.

[0058] The longitudinal positioning feedback method described in this embodiment can achieve longitudinal positioning at a position far away from the carrier substrate while ensuring the positioning accuracy in the optical tweezers experiment, thereby completing the scanning and detection of deep samples.

[0059] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission, characterized in that: The invention comprises a beam splitter (1), an objective lens (2), an imaging lens (3), an imaging sensor (4), an immersion solution (5), a multi-layer specimen slide (7), a collecting lens (8), a signal acquisition sensor (9), a front lens (10) and a displacement control device (11), wherein the imaging sensor (4), the imaging lens (3), the beam splitter (1), the objective lens (2), the multi-layer specimen slide (7), the collecting lens (8) and the signal acquisition sensor (9) are arranged in sequence from top to bottom along the optical axis, the immersion solution (5) is located above the multi-layer specimen slide (7), and the fluorescent sample (6 ) is suspended in the immersion solution (5), the front lens (10) is arranged on one side of the spectroscope (1), and the front lens (10) is connected to the displacement control device (11), the displacement control device (11) can adjust the position of the front lens (10), the laser used to capture the fluorescent sample is expanded by the front lens (10), reflected by the spectroscope (1), and then focused by the objective lens (2) onto the top of the multilayer slide (7) and captures the fluorescent sample (6) in the immersion solution, and the light emitted by the fluorescent sample (6) is emitted downward at different angles through the multilayer slide (7).

2. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission according to claim 1, characterized in that: The displacement control device (11) is connected to the acquisition sensor (9), and the displacement control device (11) changes the position of the front lens (10) according to the signal received by the signal acquisition sensor (9).

3. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission according to claim 1, characterized in that: The multilayer slide (7) comprises a polymer layer, a metal layer and a dielectric layer arranged from top to bottom.

4. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission according to claim 3 is characterized in that: The polymer layer is a polymethyl methacrylate layer, and the thickness thereof is 5 nm to 10 nm.

5. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission according to claim 3, characterized in that: The dielectric layer has a nanometer-level thickness and is composed of dielectric materials with different refractive indices.

6. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission according to claim 1, characterized in that: The signal acquisition sensor (9) is provided with a high-pass filter, which is used to filter out laser light.

7. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling emission according to claim 1, characterized in that: A narrow-band filter is provided at the entrance of the signal collection sensor (9) to ensure that the collected signal is monochromatic fluorescence.

8. A longitudinal positioning feedback method using the optical tweezers longitudinal positioning feedback device based on fluorescence coupled emission as described in any one of claims 1 to 7, characterized in that: The following steps are involved: Step 1: preparing a corresponding multilayer slide (7) according to the selected fluorescent sample (6), and dropping an immersion solution (5) containing the fluorescent sample (6) onto the prepared multilayer slide (7); Step 2: Observe the sample and the laser in the imaging sensor (4), adjust the beam splitter (1) and the objective lens (2), the laser used to capture the fluorescent sample is expanded by the front lens (10), reflected by the beam splitter (1), and then focused by the objective lens (2) to the top of the multilayer slide (7) and capture the fluorescent sample (6) in the immersion solution, and the position of the laser focus point is recorded. The light emitted by the fluorescent sample (6) is emitted downward at different angles through the multilayer slide (7). Due to the modulation of the multilayer slide (7), the radiated fluorescence will be mainly concentrated in several different angle regions, and the angle corresponding to the weakest point of fluorescence radiation between different angle regions is taken as the demarcation angle; Step three: collect the fluorescence inside and outside the boundary angle through the collecting lens (8) and the signal collection sensor (9), measure the ratio of the fluorescence intensity collected in different angle regions, calculate the fluorescence radiation intensity of the dipoles at different depths in each angle region based on the structure of the prepared multilayer slide (7) and the dipole radiation theory, and obtain the relationship between the radiation fluorescence intensity ratio and the longitudinal depth. Then, according to the measured fluorescence intensity ratio, obtain the longitudinal position of the fluorescent sample (6) in the immersion solution (5), and feed back the signal to the displacement control device (11), thereby changing the position of the front lens (10), and repeating this process to complete the precise positioning of the longitudinal position.

9. The method according to claim 8, characterized in that The preparation method of the multilayer slide (7) in step 1 is: alternately depositing dielectric layers with different refractive indices on a transparent substrate, then evaporating a metal layer, then spin coating a PMMA polymer layer, and finally drying.

10. The method according to claim 8, characterized in that The demarcation angle in step 3 is selected at different angles according to the different structures of the multilayer slide (7).

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