CN110631992A - Optical tweezers longitudinal positioning feedback device and method based on fluorescence coupling emergence - Google Patents
Optical tweezers longitudinal positioning feedback device and method based on fluorescence coupling emergence Download PDFInfo
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Abstract
The invention discloses a device and a method for feeding back optical tweezers longitudinal positioning based on fluorescence coupling emergence, wherein the device comprises: the device comprises a spectroscope, an objective lens, an imaging sensor, an immersion solution, a fluorescent sample, a multilayer slide, a collecting lens, a signal acquisition sensor, a front lens and a displacement control device. The method comprises the following steps: preparing a multilayer slide with a mode coupling and emitting function; laser used for capturing a sample is expanded and then reflected by a spectroscope and an objective lens to be focused on a fluorescent sample in an immersion solution; light emitted by the fluorescent sample is emitted downwards at different angles through the multilayer slide glass; the collected lens focuses on the signal acquisition sensor and then calculates the emergent light intensity ratio among different angle areas of the signal acquisition sensor; and calculating the longitudinal position of the fluorescent sample according to the ratio, providing a position criterion for the device, and controlling the position of the front lens through the displacement control device. The invention solves the technical problem of low longitudinal positioning accuracy of the fluorescence sample in the traditional optical tweezers system.
Description
Technical Field
The invention relates to the technical field of imaging and optical tweezers operation, in particular to a device and a method for feeding back optical tweezers longitudinal positioning based on fluorescence coupling emergence.
Background
Optical tweezers are generally a technique that uses the optical pressure in tightly focused laser to create scattering and gradient forces and to capture and manipulate microscopic particles and cells. The characteristics of non-contact, no damage and the like have great application potential in biomedicine and microscopic imaging, and the development of multiple disciplines and interdisciplinary is greatly promoted. As a far-field method, it can design, manipulate and fine-tune the light beam with nanometer precision by advanced machining means and precise mechanical control, but the positioning of the captured nanoparticles is still inaccurate, which only provides an effective control means and is difficult to reflect the specific position of the nanoparticles after the light beam acts on the particles. And due to the complex diversity of the sample, the designed light beam generates certain errors after various types of scattering. Because of this, the optical tweezers technology used in photophysics and biomedicine often cannot timely obtain the feedback of the capture effect, so that the micromanipulation technology has been difficult to be widely applied in real use. Conventional approaches to such problems often require more time and complex equipment to increase imaging resolution, using super-resolution imaging techniques to further localize the sample. How to efficiently and quickly position the sample and dynamically adjust the beam has been a problem to be solved.
For the dynamic regulation and control of light beams in the optical tweezers, most of the common implementation methods utilize a high-sensitivity positioning platform and match the existing commercial microscope. Based on the principle of optical tweezers, the force applied by a focused laser beam to particles is divided into two parts, and the axial optical force along the propagation direction of the light beam generally controls the nanoparticles to be near a substrate; while the radial optical force in the direction perpendicular to the axis of the beam, which generally has a gaussian distribution of intensity and is attractive, directs the small particles toward the optical axis and confines them near the center of the optical axis. That is, the radial component of the optical force can effectively confine the fine particles in a two-dimensional area, so that the position of the fine particles in the two-dimensional area can be well controlled by adjusting the intensity of the laser beam. Even though the full width at half maximum can not break through the diffraction limit, the modulation depth is in direct proportion to the total power of the light beam, so that the required transverse positioning precision can be improved by adjusting the power of the light beam, and theoretically can be reduced to a few nanometers. But the control and adjustment can only be carried out in the plane, and the longitudinal high-resolution information feedback cannot be realized. Meanwhile, the required platform facilities are expensive and difficult to expand, and the sample test at the laboratory stage is limited. And computer image analysis and recognition technology is used for tracing and restoring the fuzzy sample nowadays. It can only be optimized according to the existing imaging and does not really realize the effective positioning of the longitudinal position completely and effectively. Therefore, the conventional methods have certain limitations at present, which are not favorable for the technicians in the field to better apply the optical tweezers technology to biological, medical and experimental applications.
Disclosure of Invention
The invention provides a fluorescence coupling emergent-based optical tweezers longitudinal positioning feedback device and method to solve the problem that the positioning precision of the longitudinal position of a sample is not high when a light beam is dynamically regulated and controlled in the traditional optical tweezers technology.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a longitudinal positioning feedback device of optical tweezers based on fluorescence coupling exit comprises a spectroscope, an objective lens, an imaging sensor, an immersion solution, a multilayer objective plate, a collecting lens, a signal collecting sensor, a front lens and a displacement control device, wherein the imaging sensor, the imaging lens, the spectroscope, the objective lens, the multilayer objective plate, the collecting lens and the signal collecting sensor are sequentially arranged along an optical axis from top to bottom, the immersion solution is positioned above the multilayer objective plate, a fluorescent sample suspends in the immersion solution, the front lens is arranged on one side of the spectroscope and is connected with the displacement control device, the displacement control device can adjust the position of the front lens, and laser used for capturing the fluorescent sample is reflected by the spectroscope after being expanded by the front lens and then is focused above the multilayer objective plate by the objective lens and captures the fluorescent sample in the immersion solution, light emitted by the fluorescent sample is emitted downward at different angles through the multi-layer slide.
Furthermore, the displacement control device is connected with 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.
Further, the multilayer slide comprises a polymer layer, a metal layer and a dielectric layer which are arranged from top to bottom.
Further, the polymer layer is a polymethyl methacrylate layer, and the thickness of the polymer layer is 5nm to 10 nm.
Further, the dielectric layer is of nanometer-scale thickness and is made of dielectric materials with different refractive indexes.
Furthermore, a high-pass filter is arranged in the signal acquisition sensor and used for filtering laser.
Furthermore, a narrow-band filter is arranged at the inlet of the signal acquisition sensor, so that the collected signals are guaranteed to be monochromatic fluorescence.
The longitudinal positioning feedback method adopting the optical tweezers longitudinal positioning feedback device based on fluorescence coupling emergence comprises the following steps:
the method comprises the following steps: preparing a corresponding multilayer slide according to the selected fluorescent sample, and dripping the immersion solution containing the fluorescent sample on the prepared multilayer slide;
step two: observing a sample and laser in an imaging sensor, adjusting a spectroscope and an objective lens, wherein the laser used for capturing a fluorescent sample is reflected by the spectroscope after being expanded by a front lens, then is focused above a multilayer objective lens through the objective lens, captures the fluorescent sample in an immersion solution, and records the position of a laser focusing point;
step three: the fluorescence inside and outside the boundary angle is collected through the collecting lens and the signal collecting sensor, the fluorescence intensity ratio collected in different angle areas is measured, the fluorescence radiation intensity of dipoles with different depths in each angle area is calculated according to the structure of the prepared multilayer slide and a dipole radiation theory, the relation between the radiation fluorescence intensity ratio and the longitudinal depth is obtained, the longitudinal position of a fluorescence sample in an immersion solution is obtained according to the measured fluorescence intensity ratio, signals are fed back to the displacement control device, the position of the front lens is changed, and the process is repeated to complete the accurate positioning of the longitudinal position.
Further, the preparation method of the multilayer slide in the first step comprises the following steps: alternately depositing dielectric layers with different refractive indexes on a transparent substrate, then evaporating a metal layer, spin-coating a PMMA polymer layer, and finally drying.
Further, the dividing angle in step three is selected to be different according to the structure of the multilayer slide.
In the invention, in the traditional optical tweezers technology, the glass slide is replaced by the multilayer slide, and the fluorescence collecting device is additionally arranged below the multilayer slide, so that the system can collect fluorescence coupling emergent signals while the optical tweezers capture the signals, and further the intensity ratios of different coupling emergent modes are measured. The longitudinal position of the fluorescence sample is accurately calculated and fed back to the electric control precision displacement table by combining the designed multilayer slide structure and the change of the light beam in the fine adjustment process, thereby realizing the precise dynamic feedback in the optical tweezers system.
Compared with the prior art, the invention has the advantages that:
(1) high-precision longitudinal positioning: the multilayer slide glass is introduced in the traditional optical tweezers technology, the optical tweezers system is expanded, a new calibration feedback means is provided, the coupling emergent intensity can be measured by using a collecting device while a fluorescence sample is observed and captured, and high-precision longitudinal positioning can be carried out at any absolute position in an immersion solution by combining the structural characteristics of the multilayer slide glass;
(2) simple structure, low cost: the observation of longitudinal micro displacement can be realized only by utilizing the inherent multilayer structure of multilayer slide glass and a simple collecting device without complex confocal components in a commercial microscopic system and special fluorescent materials required in super-resolution imaging, and signals are fed back to the front lens to adjust light beams in time;
(3) easy to expand, the application range is wide: the optical tweezers longitudinal positioning feedback device based on fluorescence coupling emergence is easy to attach to the existing optical tweezers device, can realize real-time observation of the longitudinal positions of various fluorescence samples in the traditional light beam binding and light capturing, can conveniently and dynamically regulate and control the light beam positions in an optical tweezers system, and improves the existing sensing precision.
Drawings
Fig. 1 is a schematic structural diagram of an optical tweezers longitudinal positioning feedback device based on fluorescence coupling and emission according to the present invention.
FIG. 2 is a graph of the calculated longitudinal position versus the outside-inside ratio of the cut-off angle for the multilayer slide configuration of the example embodiment.
Fig. 3 is a flowchart of the third step of the optical tweezers longitudinal positioning feedback method based on fluorescence coupling and emission according to the present invention.
The reference numbers in the figures are: 1. the device comprises a spectroscope, 2, an objective lens, 3, an imaging lens, 4, an imaging sensor, 5, an immersion solution, 6, a fluorescent sample, 7, a multilayer slide, 8, a collecting lens, 9, a signal acquisition sensor, 10, a front lens, 11 and a displacement control device.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 1, a longitudinal positioning feedback device for optical tweezers based on fluorescence coupling exit comprises a spectroscope 1, an objective lens 2, an imaging lens 3, an imaging sensor 4, an immersion solution 5, a multilayer objective plate 7, a collecting lens 8, a signal collecting sensor 9, a front lens 10 and a displacement control device 11, wherein the imaging sensor 4, the imaging lens 3, the spectroscope 1, the objective lens 2, the multilayer objective plate 7, the collecting lens 8 and the signal collecting sensor 9 are sequentially arranged along an optical axis from top to bottom, the immersion solution 5 is arranged above the multilayer objective plate 7, a fluorescence sample 6 is suspended in the immersion solution 5, the front lens 10 is arranged on one side of the spectroscope 1, the front lens 10 is connected with the displacement control device 11, and the displacement control device can control the position of the front lens 10. The multilayer slide 7 is preferably a multilayer slide, the displacement control device 11 is preferably an electrically controlled precision displacement stage, and is used for capturing the fluorescent sample 6, wherein the laser light is expanded by the front lens 10, reflected by the spectroscope 1 and focused on the multilayer slide 7 through the objective lens 2, and capturing the fluorescent sample 6 in the immersion solution, and the light emitted by the fluorescent sample 6 is emitted downwards at different angles through the multilayer slide 7.
The displacement control device 11 is connected with 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.
The multilayer slide 7 comprises a polymer layer, a metal layer and a dielectric layer which are arranged from top to bottom. Wherein the polymer layer is preferably a polymethyl methacrylate layer, and the thickness of the polymer layer is 5nm to 10 nm; the thickness of the metal layer is 40nm to 50 nm. The dielectric layer is of nanometer-level thickness and is made of dielectric materials with different refractive indexes. Therefore, the fluorescent light emitted by the fluorescent sample is partially emitted within the boundary angle and partially emitted outside the boundary angle.
The fluorescent sample may be located anywhere in the immersion solution. And when the fluorescence sample is positioned at different longitudinal positions in the immersion solution, the intensity ratio of the fluorescence collected by the signal acquisition sensor in the boundary angle is different from that of the fluorescence collected by the signal acquisition sensor outside the boundary angle.
And a high-pass filter is arranged in the signal acquisition sensor 9 and used for filtering laser. Ensures that the collected optical signals are pure fluorescence signals.
A narrow-band filter is arranged at the inlet of the signal acquisition sensor 9 to ensure that the collected signal is monochromatic fluorescence.
The numerical aperture of the collecting lens is large enough to complete the collection of the fluorescence inside and outside the boundary angle. Alternatively, the collecting lens may be replaced by an objective lens to achieve collection of the fluorescence inside and outside the cut-off angle.
The longitudinal positioning feedback method of the optical tweezers longitudinal positioning feedback device based on fluorescence coupling emergence specifically comprises the following steps:
the method comprises the following steps: preparing a corresponding multilayer slide 7 according to the selected fluorescent sample 6, and dripping the immersion solution 5 containing the fluorescent sample 6 on the prepared multilayer slide 7;
step two: observing a sample and laser in an imaging sensor 4, adjusting a spectroscope 1 and an objective lens 2, reflecting the laser used for capturing a fluorescent sample by the spectroscope 1 after being expanded by a front lens 10, focusing the laser above a multilayer objective plate 7 by the spectroscope 1 and capturing the fluorescent sample 6 in an immersion solution by the objective lens 2, and recording the position of a laser focusing point, wherein the light emitted by the fluorescent sample 6 is emitted downwards at different angles by the multilayer objective plate 7, due to the modulation of the multilayer objective plate 7, the radiated fluorescence is mainly concentrated in a plurality of different angle areas, and the angle corresponding to the weakest part of the fluorescent radiation between the different angle areas is taken as a boundary angle;
step three: the fluorescence inside and outside the boundary angle is collected through the collecting lens 8 and the signal collecting sensor 9, the ratio of the collected fluorescence intensity in different angle areas is measured, the fluorescence radiation intensity of dipoles with different depths in each angle area is calculated according to the structure of the prepared multilayer slide 7 and the dipole radiation theory, the relation between the ratio of the radiation fluorescence intensity and the longitudinal depth is obtained, the longitudinal position of the fluorescence sample 6 in the immersion solution 5 is obtained according to the measured fluorescence intensity ratio, the signal is fed back to the displacement control device 11, the position of the front lens 10 is changed, and the process is repeated, so that the precise positioning of the longitudinal position is completed.
The preparation method of the multilayer slide 7 in the first step comprises the following steps: alternately depositing dielectric layers with different refractive indexes on a transparent substrate, then evaporating a metal layer, spin-coating a polymer layer, and finally drying.
The dividing angle in step three is selected to be different according to the structure of the multilayer slide 7.
In the third step, the precise positioning of the longitudinal position of the fluorescence sample in the immersion solution needs to be determined by combining the laser focusing surface observed during the rough adjustment and the measured fluorescence intensity ratio. And determining the approximate position range of the fluorescent sample according to the focal plane position, finely adjusting the front lens by an electric control precision displacement table to enable the light beam to be finely focused up and down, and judging the movement direction and the precise position of the fluorescent sample by utilizing the change trend of the fluorescence intensity ratio. Thereby feeding back signals to the electric control precision displacement table again to finish the precise dynamic regulation and control of the fluorescence sample in the optical tweezers experiment.
Example 1
A longitudinal positioning feedback device of optical tweezers based on fluorescence coupling emergence comprises a spectroscope 1, an objective lens 2, an imaging lens 3, an imaging sensor 4, an immersion solution 5, a fluorescence sample 6, a multilayer slide glass 7, a collecting lens 8, a signal collecting sensor 9, a front lens 10 and an electric control precision displacement table 11. Imaging sensor 4, imaging lens 3, spectroscope 1, objective 2, multilayer slide glass, collection lens 8 and signal acquisition sensor 9 set gradually along the optical axis, multilayer slide glass 7 top is arranged in to immersion solution 5, and fluorescence sample 6 suspends in immersion solution 5, leading lens 10 is connected to automatically controlled accurate displacement platform 11 to the position of leading lens 10 is controlled.
Further, the multilayer slide 7 comprises a polymer layer, a metal layer and a dielectric layer arranged from top to bottom. The thickness of the medium layer of the multilayer slide 7 can be changed according to the fluorescent sample 6 to be tested.
Further, the polymer layer of the multilayer slide 7 is a polymethyl methacrylate layer having a thickness of 10 nm.
Further, the metal layer material of the multilayer slide 7 is silver and has a thickness of 40 nm.
Further, the dielectric layers of the multilayer slide glass 7 are alternating silicon nitride layers and silicon dioxide layers, and the thickness of each layer is not more than 200 nm.
Further, the front lens 10 can change its position according to the signal received by the electrically controlled precision displacement stage. It has been set that the high level signal is shifted to the right (away from the beam splitter 1) and the low level signal is shifted to the left (towards the beam splitter 1).
Further, the immersion solution 5 is an aqueous solution, and the fluorescent sample 6 is suspended in the immersion solution 5. When the fluorescence samples 6 are positioned at different longitudinal positions in the immersion solution 5, the ratio of the fluorescence collected by the signal acquisition sensor 9 is different between the inside and the outside of the boundary angle.
Further, the signal acquisition sensor 9 comprises a high-pass filter for filtering the collected laser. Additionally, a narrow-band filter is added in front of the signal acquisition sensor 9 to ensure that the collected signal is monochromatic fluorescence.
Further, the collecting lens 8 is selected as an objective lens, and can collect the fluorescence inside and outside the boundary angle.
By adopting the longitudinal positioning feedback method of the optical tweezers longitudinal positioning feedback device based on fluorescence coupling and emission, in this embodiment, the sample is positioned to a position 200nm above the multilayer slide. The method comprises the following steps:
(1) preparing a multilayer slide glass according to the selected fluorescent sample 6 as the rhodamine 6G doped fluorescent bead, wherein the preparation of the multilayer slide glass comprises the following steps: alternately depositing silicon nitride and silicon dioxide on a transparent cover glass, then evaporating a metal layer, finally spin-coating a PMMA polymer layer, drying, and then dripping an immersion solution 5 containing a fluorescent sample 6 on the upper part of the multilayer slide glass;
(2) observing in an imaging sensor 4, adjusting a spectroscope 1 to focus a laser beam near the upper part of the multilayer slide glass and capture a fluorescent sample 6, and determining the position of a laser focal plane, wherein the laser focal plane is positioned near the 200nm position above the multilayer slide glass;
(3) collecting the fluorescence inside and outside the boundary angle through the collecting lens 8 and the signal collecting sensor 9, taking the boundary angle as 58 degrees, measuring the initial ratio of the collected fluorescence intensity inside and outside the boundary angle as 12.5, and calculating the ratio of the fluorescence intensity inside and outside the boundary angle as 11.93 when the fluorescence sample is positioned in the immersion solution at a distance of 200nm from the multilayer slide glass according to the structure of the multilayer slide glass 7 prepared in the step one, wherein the ratio is defined as a reference value, as shown in fig. 2. Firstly, the electric control precision displacement platform obtains a high level signal, and then an initial value signal is fed back to the electric control precision displacement platform, so that the position of the front lens 10 is adjusted to move right, and the position of a focused light beam is changed. The collected fluorescence intensity ratio is again recorded and calculated and the position of the front lens is again automatically feedback adjusted until the difference between the measured intensity ratio and the reference value is less than 0.01. Thereby completing the accurate positioning of the longitudinal position, as shown in fig. 3.
The method records different emergent intensities of different angles in the sensor, and utilizes the ratio to finish the accurate positioning of the position of the captured sample, and the error theory is less than 16 nm.
The longitudinal positioning feedback method described in this embodiment can complete longitudinal positioning at a position far from the object carrying substrate while ensuring the positioning accuracy in the optical tweezers experiment, and complete scanning and detection of a deep layer sample.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. The longitudinal positioning feedback device for the optical tweezers based on fluorescence coupling exit is characterized by comprising a spectroscope (1), an objective lens (2), an imaging lens (3), an imaging sensor (4), an immersion solution (5), a multilayer objective lens (7), a collecting lens (8), a signal collecting sensor (9), a front lens (10) and a displacement control device (11), wherein the imaging sensor (4), the imaging lens (3), the spectroscope (1), the objective lens (2), the multilayer objective lens (7), the collecting lens (8) and the signal collecting sensor (9) are sequentially arranged along an optical axis from top to bottom, the immersion solution (5) is positioned above the multilayer objective lens (7), a fluorescence 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 with the displacement control device (11), the displacement control device (11) can adjust the position of the front lens (10), laser used for capturing a fluorescent sample is reflected by the spectroscope (1) after being expanded by the front lens (10) and then focused above the multilayer objective (7) through the objective (2) and captures the fluorescent sample (6) in the immersion solution, and light emitted by the fluorescent sample (6) is emitted downwards at different angles through the multilayer objective (7).
2. The optical tweezers longitudinal positioning feedback device based on fluorescence coupling exit according to claim 1, wherein the displacement control device (11) is connected to the collection sensor (9), and the displacement control device (11) changes the position of the front lens (10) according to the signal received by the signal collection sensor (9).
3. The fluorescence coupling extraction-based optical tweezers longitudinal positioning feedback device according to claim 1, wherein the multilayer slide (7) comprises a polymer layer, a metal layer and a dielectric layer arranged from top to bottom.
4. The fluorescence coupling extraction-based optical tweezers longitudinal positioning feedback device of claim 3, wherein the polymer layer is a polymethyl methacrylate layer with a thickness of 5nm to 10 nm.
5. The fluorescence coupling extraction-based optical tweezers longitudinal positioning feedback device of claim 3, wherein the dielectric layer is of nanometer-scale thickness and is made of dielectric materials with different refractive indexes.
6. The fluorescence coupling emission-based optical tweezers longitudinal positioning feedback device according to claim 1, wherein a high-pass filter is arranged in the signal acquisition sensor (9), and the high-pass filter is used for filtering laser.
7. The fluorescence coupling extraction based optical tweezers longitudinal positioning feedback device according to claim 1, wherein a narrow band filter is arranged at the entrance of the signal collection sensor (9) to ensure that the collected signal is monochromatic fluorescence.
8. Longitudinal positioning feedback method using the fluorescence coupling extraction based optical tweezers longitudinal positioning feedback device according to any one of claims 1 to 7, comprising the following steps:
the method comprises the following steps: preparing a corresponding multilayer slide (7) according to the selected fluorescent sample (6), and dripping the immersion solution (5) containing the fluorescent sample (6) on the prepared multilayer slide (7);
step two: observing a sample and laser in an imaging sensor (4), adjusting a spectroscope (1) and an objective lens (2), focusing the laser used for capturing a fluorescent sample above a multilayer objective plate (7) through the spectroscope (1) after being expanded by a front lens (10) and then focusing the laser above the multilayer objective plate (2) and capturing a fluorescent sample (6) in an immersion solution, and recording the position of a laser focusing point, wherein the light emitted by the fluorescent sample (6) is emitted downwards at different angles through the multilayer objective plate (7), due to the modulation of the multilayer objective plate (7), the radiated fluorescence is mainly concentrated in a plurality of different angle areas, and the angle corresponding to the weakest position of the fluorescent radiation among the different angle areas is taken as a boundary angle;
step three: fluorescence inside and outside the boundary angle is collected through a collecting lens (8) and a signal collecting sensor (9), the fluorescence intensity ratio collected in different angle areas is measured, the fluorescence radiation intensity of dipoles with different depths in each angle area is calculated according to the structure of the prepared multilayer slide (7) and a dipole radiation theory, the relation between the radiation fluorescence intensity ratio and the longitudinal depth is obtained, the longitudinal position of a fluorescence sample (6) in an immersion solution (5) is obtained according to the measured fluorescence intensity ratio, a signal is fed back to a displacement control device (11), the position of a front lens (10) is changed, and the process is repeated to complete accurate positioning of the longitudinal position.
9. The method according to claim 8, wherein the multilayer slide (7) is prepared in step one by: alternately depositing dielectric layers with different refractive indexes on a transparent substrate, then evaporating a metal layer, spin-coating a PMMA polymer layer, and finally drying.
10. The method according to claim 8, characterized in that the dividing angle in step three is selected to be different depending on the structure of the multilayer slide (7).
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