Super-resolution laser direct writing and imaging method and device based on two-photon and stimulated emission depletion effect

Abstract

A super-resolution laser direct writing and imaging method and device based on two-photon and stimulated emission depletion effects, the method comprising: adding a photoinitiator to the photoresist, utilizing its two-photon absorption effect and stimulated emission depletion effect to realize two-photon STED high-precision micro-nanostructure laser direct writing; at the same time, utilizing its own fluorescent luminescence characteristics, utilizing its two-photon absorption characteristics and stimulated emission depletion (STED) characteristics, to perform two-photon STED super-resolution microscopic imaging of the written structure. The present invention utilizes the two-photon and STED effects of the initiator to simultaneously realize super-resolution writing and imaging, simplifying the composition of the photoresist. In addition, the same excitation light and loss light can be used in writing and imaging, respectively, so high-precision writing and super-resolution imaging can be realized simultaneously in one optical system, and the system complexity can be effectively simplified by multiplexing the excitation light and loss light optical paths of writing and imaging.

Description

Technical Field

[0001] The present invention relates to the field of ultra-precision laser direct writing and super-resolution microscopic imaging, and in particular to a super-resolution laser direct writing and imaging method and device based on two-photon and stimulated emission depletion effects. Background Art

[0002] With the invention of femtosecond lasers, nonlinear properties in materials have been discovered and utilized one after another. In recent years, micro-nanostructure processing based on the nonlinear interaction between light and matter induced by femtosecond pulse lasers has made great progress and has shown bright prospects in science, technology and various industries. In laser direct writing, compared with the traditional single-photon absorption-induced polymerization, the two-photon or multi-photon properties of materials can improve the writing resolution to sub-hundred nanometers and enable it to have the processing capability of any three-dimensional structure. However, to break through the writing resolution of sub-50 nanometers, it is not enough to use only the two-photon effect. In recent years, inspired by STED super-resolution microscopy, a certain photoinitiator, such as DETC, ITX, etc., is added to the equipped photoresist, and another beam of light is introduced as an inhibitory light at the edge of the writing excitation light to eliminate the polymerization effect caused by the two-photon excitation light at the edge of the photoresist, which can further improve the resolution to sub-50 nanometers and effectively reduce the critical effect of writing.

[0003] In order to characterize the high-precision structure of the writing, the traditional method is usually to develop the writing structure, then spray gold, and then observe with an electron microscope or an atomic force microscope. However, due to the slow imaging speed of the electron microscope, the environmental requirements are high, and the cost is high. In addition, adding specific dyes to the photoresist material for marking to achieve optical super-resolution imaging has also been reported, but the addition of dyes will inevitably have an unmeasurable impact on the chemical properties of the photoresist itself, thereby affecting the writing quality. In order to solve the above problems, the present invention only utilizes the fluorescent luminescence characteristics of the photoinitiator added in the photoresist for initiating writing polymerization, and in particular, utilizes its two-photon absorption characteristics and stimulated radiation loss characteristics, and regulates the luminescence of the loss structure edge by lateral and axial light spots, so that lateral and axial three-dimensional super-resolution imaging can be achieved. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and also to expand the prior art, and to provide a super-resolution laser direct writing and imaging method and device based on two-photon and stimulated emission depletion effects.

[0005] The present invention utilizes the two-photon and stimulated depletion effects of the initiator in the photoresist to achieve two functions at the same time: first, the photoresist is triggered to generate two-photon polymerization, and a beam of hollow inhibition light is introduced at the edge of the excitation light to inhibit edge polymerization, thereby realizing super-resolution laser direct writing of high-precision micro-nano structures; at the same time, there is no need to additionally dope dyes in the photoresist, but the fluorescence luminescence characteristics of the initiator itself are utilized to introduce a beam of hollow loss light at the edge of the two-photon excitation generated by the excitation light, and the fluorescence generated by the two-photon excitation at the loss edge is lost, thereby realizing super-resolution microscopic imaging characterization of the written structure. The excitation light used to trigger the photoresist to generate two-photon polymerization and the inhibition light to inhibit its polymerization can be the same light beam as the excitation light used to generate two-photon excitation in imaging and the loss light to lose its luminescence, so high-precision writing and super-resolution imaging can be realized simultaneously in one optical system, and the system complexity can be effectively simplified by multiplexing the excitation light and loss light optical paths for writing and imaging.

[0006] The first aspect of the present invention relates to a super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effects, comprising the following steps:

[0007] (1) Preparation of photoresist: adding photoinitiator to photoresist monomer;

[0008] (2) Laser direct writing based on two-photon and stimulated emission depletion effects: Two laser beams are focused on the photoresist through an objective lens. One beam is the excitation light, which forms a solid spot to induce two-photon polymerization of the photoresist. The other beam is the inhibition light, which forms a hollow spot to inhibit the polymerization of the photoresist. By aligning the center of the excitation solid light spot with the center of the inhibition hollow light spot, super-resolution laser direct writing of high-precision micro-nano structures can be achieved.

[0009] (3) Development: Develop the engraved structure and air dry it;

[0010] (4) Two-photon STED super-resolution microscopy based on two-photon and stimulated emission depletion effects: The air-dried inscribed structure is placed back on the sample stage, and two laser beams are focused on the sample through an objective lens. One beam is the excitation light, which forms a solid spot to excite the initiator to produce two-photon effect and emit fluorescence. The other beam is the loss light, which forms a hollow spot. The fluorescence generated at the edge of the loss structure can be overlapped with the center of the excitation light and the loss light to achieve two-photon STED super-resolution microscopy of the inscribed structure.

[0011] Preferably, the writing process can adopt two modes: the excitation light and the suppression light pass through the objective lens, the oil, and the substrate, and then converge on the photoresist dripped on the substrate for writing, or the excitation light and the suppression light pass through the objective lens and directly converge on the photoresist between the upper part of the objective lens and the substrate for writing;

[0012] Preferably, the imaging process can adopt two modes: the excitation light and the loss light pass through the objective lens, the oil, and the substrate, and converge to the inscribed structure on the upper surface of the substrate for two-photon STED imaging, or pass through the objective lens, the oil, and the substrate, and converge to the inscribed structure immersed in the lower surface of the substrate for two-photon STED imaging;

[0013] Preferably, the initiator may be a material with high luminescence efficiency such as 7-diethylamino-3-(2-thienyl)coumarin (DETC);

[0014] Preferably, the ratio of DETC to the total weight of the mixture of the photoresist material monomer and DETC is 1%.

[0015] The second aspect of the present invention relates to a device for implementing a laser direct writing and imaging method based on two-photon and stimulated depletion effect of the present invention, including an excitation optical path for inducing two-photon polymerization of photoresist; an inhibition optical path for inhibiting polymerization of photoresist; a combined optical path of the excitation optical path and the inhibition optical path for writing; an excitation optical path for performing two-photon excitation on an initiator in an inscribed structure to generate fluorescence; a loss optical path for depleting the initiator to generate fluorescence; a combined optical path of the excitation optical path and the loss optical path for imaging; a fluorescence imaging detection optical path; a high-precision piezoelectric three-dimensional displacement stage for performing two-photon STED super-resolution microscopy imaging on the inscribed structure; an excitation optical path and a loss optical path for two-photon STED super-resolution microscopy imaging;

[0016] The excitation optical path for inducing two-photon polymerization of the photoresist is sequentially provided with a first laser, a pulse pre-compression module, a first acousto-optic modulator, a first objective lens, a first pinhole, a first lens, and a first half-wave plate;

[0017] The suppression optical path for suppressing the two-photon polymerization of the photoresist is provided with a second laser, a second acousto-optic modulator, a second objective lens, a second pinhole, a second lens, a first reflector, and a second half-wave plate in sequence;

[0018] The combined beam path of the writing excitation light and the inhibition light is sequentially provided with a polarization beam splitter, a third half-wave plate, a spatial light modulator, a fourth half-wave plate, a quarter-wave plate, a dichroic mirror, a scanning galvanometer, a scanning lens, a second reflecting mirror, a field lens, a third objective lens, a high-precision piezoelectric three-dimensional displacement stage, a sample holder, a substrate, and a photoresist sample containing an initiator;

[0019] The excitation light path and loss light path of the imaging are the same as the excitation light path and suppression light path of the writing;

[0020] The combined beam path of the excitation light path and the loss light path for imaging and the combined beam path of the excitation light path and the suppression light path for writing are the same light path;

[0021] The fluorescence imaging detection optical path is sequentially provided with a writing sample, a substrate, a sample holder, a high-precision piezoelectric three-dimensional displacement stage, a third objective lens, a field lens, a second reflector, a scanning lens, a scanning galvanometer, a dichroic mirror, a third reflector, a filter, a third lens, a multimode optical fiber, and an avalanche diode photodetector;

[0022] It also includes a computer connected to the first acousto-optic modulator, the second acousto-optic modulator, the spatial light modulator, the scanning galvanometer, the high-precision piezoelectric three-dimensional translation stage and the avalanche diode photodetector;

[0023] The laser emitted by the first laser is used as the excitation light for inducing the photoresist to produce two-photon polymerization. The excitation light passes through the dispersion pre-compensation module to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The laser after the dispersion compensation passes through the first acousto-optic modulator to control its intensity and switch, and then converges through the first objective lens, and then passes through the first pinhole for spatial filtering to filter out the influence of the preceding optical elements on the quality of the light spot. The first pinhole is located at the focal plane of the first objective lens. The filtered light beam passes through the first lens for collimation and expansion to ensure that the beam diameter after expansion can fill the entrance pupil of the last oil-immersed third objective lens. The expanded laser passes through the first half-wave plate to adjust its linear polarization direction to vertical polarization;

[0024] The laser light emitted by the second laser is used as the inhibiting light for inhibiting the polymerization of the photoresist. The inhibiting light is controlled in intensity and on and off by the second acousto-optic modulator, then converged by the second objective lens, and then spatially filtered by the second pinhole to filter out the influence of the preceding optical element on the quality of the light spot. The second pinhole is located at the focal plane of the second objective lens. The filtered light beam is collimated and expanded by the second lens to ensure that the diameter of the expanded light beam can fill the entrance pupil of the third objective lens that is finally immersed in oil. The expanded inhibiting light is reflected by the first reflector, and then passes through the second half-wave plate to adjust its polarization direction to horizontal polarization.

[0025] The excitation light is reflected by the polarization beam combiner, and the suppression light is transmitted through the polarization beam combiner to realize the combination of the excitation light and the suppression light. The combined light passes through the third half-wave plate and then passes through the spatial light modulator to change the fast and slow axes of the third half-wave plate, and adjust the polarization direction of the suppression light to be consistent with the polarization direction of the spatial light modulator. The spatial light modulator is divided into left and right screens. The left screen loads the corresponding phase to modulate the excitation light into solid light and adjusts the light spot aberration to the minimum. The right screen loads the corresponding phase to modulate the suppression light into hollow light, or cooperates with adjusting the fast and slow axis direction of the third half-wave plate. The left and right screens adjust the light spot in turn, that is, the left screen adjusts the suppression light to be hollow light, and the right screen adjusts the light spot to be hollow light. The screen adjusts the excitation light to be solid light, and the modulated light beam passes through a fourth half-wave plate and a quarter-wave plate, so that the suppression light finally passes through the third objective lens to be a hollow light spot with a dark center to the bottom, and then passes through a dichroic mirror to reflect into a scanning galvanometer to scan the excitation light and the suppression light, and then passes through a scanning lens, and then reflects through a second reflection mirror to enter a field lens, and the field lens collimates the light beam into parallel light and incidents on the third objective lens, and then focuses through the third objective lens into convergent light and incidents on a photoresist configured with an initiator to wait for writing, wherein the excitation light is a solid light spot on the sample surface, and the suppression light is a hollow light with a transverse distribution or an axial distribution that is sleeved on the edge of the excitation light;

[0026] The computer controls the first optical modulator and the second acousto-optic modulator, controls the switching of the laser and the light intensity, and controls the scanning and movement of the scanning galvanometer and the high-precision piezoelectric three-dimensional translation stage to achieve high-precision two-photon STED writing of micro-nano structures.

[0027] The excitation optical path and the loss optical path used in the two-photon STED super-resolution microscopy imaging process are the same optical path as the excitation optical path and the suppression optical path used to achieve high-precision writing;

[0028] The fluorescence emitted by the initiator in the writing structure is received by the third objective lens, then converged by the field lens, reflected by the second reflector, then passed through the scanning lens, and descanned by the scanning galvanometer, then transmitted by the dichroic mirror, then reflected by the third reflector, and then filtered out by the filter to remove stray light caused by laser and environmental factors, and then converged by the third lens. The converged light is then received and transmitted by the multimode optical fiber, and finally the fluorescence signal is detected by the avalanche diode photodetector;

[0029] The computer controls the first acousto-optic modulator and the second acousto-optic modulator to adjust the intensity and switch of the initiator excitation light, controls the scanning galvanometer to scan the light beam, and performs data processing and transmission on the received fluorescence signal to realize two-photon STED super-resolution microscopic imaging of the inscribed structure.

[0030] Preferably, the first laser is a femtosecond laser;

[0031] Preferably, the wavelength of the first laser may be between 500nm and 800nm;

[0032] Preferably, the second laser may be a continuous light laser, or a ps or ns laser;

[0033] Preferably, the second laser wavelength may be 532 nm;

[0034] Preferably, the diameter of the multimode optical fiber is selected to be the size of one Airy disk diameter after system amplification;

[0035] Preferably, the scanning galvanometer, the scanning lens, the field lens and the entrance pupil of the third objective lens form a 4f system;

[0036] Preferably, the substrate may be made of a transparent material such as a cover glass, quartz, sapphire, or a non-transparent material such as a silicon wafer.

[0037] The third aspect of the present invention relates to a method for super-resolution writing and imaging using the device of the present invention, which specifically comprises the following steps:

[0038] (1) The laser emitted by the first laser is used as the excitation light for inducing two-photon polymerization of the photoresist. The excitation light passes through the dispersion pre-compensation module to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The laser after the dispersion compensation passes through the first acousto-optic modulator to control its intensity and switch, and then passes through the first objective lens to converge, and then passes through the first pinhole to perform spatial filtering to filter out the influence of the preceding optical elements on the light spot quality. The first pinhole is located at the focal plane of the first objective lens. The filtered light beam passes through the first lens to collimate and expand the beam to ensure that the beam diameter after expansion can fill the entrance pupil of the last oil-immersed third objective lens. The expanded laser passes through the first half-wave plate to adjust its linear polarization direction to vertical polarization;

[0039] (2) The laser light emitted by the second laser is used as the inhibition light for inhibiting the polymerization of the photoresist. The inhibition light is controlled by the second acousto-optic modulator for intensity and switch, and then converged by the second objective lens, and then spatially filtered by the second pinhole to filter out the influence of the preceding optical element on the quality of the light spot. The second pinhole is located at the focal plane of the second objective lens. The filtered light beam is then collimated and expanded by the second lens to ensure that the diameter of the expanded light beam can fill the entrance pupil of the third oil-immersed objective lens. The expanded inhibition light is then reflected by the first reflector, and then passed through the second half-wave plate to adjust its polarization direction to horizontal polarization.

[0040] (3) The excitation light is reflected by the polarization beam combiner, and the suppression light is transmitted by the polarization beam combiner to realize the combination of the excitation light and the suppression light. The combined light passes through the third half-wave plate and then passes through the spatial light modulator to change the fast and slow axes of the third half-wave plate, and adjust the polarization direction of the suppression light to be consistent with the polarization direction of the spatial light modulator. The spatial light modulator is divided into left and right screens. The left screen is loaded with the corresponding phase to modulate the excitation light into solid light and adjust the light spot aberration to the minimum. The right screen is loaded with the corresponding phase to modulate the suppression light into hollow light. (Or the left and right screens adjust the light spot in reverse by adjusting the fast and slow axis directions of the third half-wave plate, that is, the left screen adjusts the suppression light into hollow light. , the right screen adjusts the excitation light to be solid light), the modulated light beam passes through the fourth half wave plate and the quarter wave plate, so that the suppression light finally passes through the third objective lens as a hollow light spot with a dark center to the bottom, and then is reflected by the dichroic mirror into the scanning galvanometer to scan the excitation light and the suppression light, and then passes through the scanning lens, and then is reflected by the second reflection mirror into the field lens, the field lens collimates the light beam into parallel light and is incident on the third objective lens, and then is focused by the third objective lens into convergent light and is incident on the photoresist configured with an initiator to wait for writing, wherein the excitation light is a solid light spot on the sample surface, and the suppression light is a hollow light with a transverse distribution or an axial distribution that is sleeved on the edge of the excitation light;

[0041] (4) There are two types of writing modes. The first is to drip oil on the third objective lens, and place a substrate (a transparent substrate such as a cover glass, quartz, etc.) on the third objective lens, and then place the substrate on a sample holder. The sample holder is placed on a high-precision piezoelectric three-dimensional displacement stage, and photoresist is dripped on the substrate. The light spot converges on the upper surface of the substrate and writes upwards. The second is an immersion writing mode, that is, dripping photoresist on the third objective lens, focusing the converged light on the lower surface of the substrate (a transparent substrate such as a cover glass, quartz, etc., or a non-transparent substrate such as a silicon wafer), and writing downwards.

[0042] (5) The first optical modulator and the second acousto-optic modulator are controlled by a computer to realize the switching of the laser and the light intensity control. The scanning galvanometer and the scanning and movement of the high-precision piezoelectric three-dimensional translation stage are controlled by a computer to realize the two-photon STED writing of high-precision micro-nano structures.

[0043] (6) The engraved micro-nanostructure is developed and then placed back on the high-precision piezoelectric three-dimensional translation stage to wait for two-photon STED super-resolution microscopy imaging of the engraved structure;

[0044] (7) The excitation optical path and the loss optical path used in the two-photon STED super-resolution microscopy imaging process are the same optical path as the excitation optical path and the suppression optical path used to achieve high-precision writing;

[0045] (8) The process of performing two-photon STED super-resolution microscopy also includes two modes: the first mode is that the excitation light and the loss light focused by the third objective lens pass through the oil above the third objective lens, and then pass through the substrate (transparent substrate such as cover glass) to converge on the writing structure on the upper surface of the substrate dripped with oil to achieve two-photon excitation and STED loss of the initiator in the writing structure; the second mode is to drip oil above the third objective lens, and place the substrate with the structure (lens or non-lens substrate) facing downward above the third objective lens, and the excitation light and the loss light focused by the third objective lens converge on the writing structure on the lower surface of the substrate to achieve two-photon excitation and STED loss;

[0046] (9) The fluorescence emitted by the initiator in the writing structure is received by the third objective lens, then converged by the field lens, reflected by the second reflector, then passed through the scanning lens, and descanned by the scanning galvanometer, then transmitted by the dichroic mirror, then reflected by the third reflector, and then filtered out by the filter to remove stray light caused by laser and environmental factors, and then converged by the third lens. The converged light is then received and transmitted by the multimode optical fiber, and finally the fluorescence signal is detected by the avalanche diode photodetector;

[0047] (10) The computer outputs control signals to control the first acousto-optic modulator and the second acousto-optic modulator to adjust the intensity of the initiator excitation light and switch it, control the scanning galvanometer to scan the light beam, and perform data processing and transmission on the received fluorescence signal, thereby realizing two-photon STED super-resolution microscopic imaging of the inscribed structure.

[0048] The present invention adds a photoinitiator to the photoresist, and utilizes its two-photon absorption effect and stimulated depletion effect to realize two-photon STED high-precision micro-nano structure laser direct writing; at the same time, by utilizing its own fluorescent luminescence characteristics, and further utilizing its two-photon absorption characteristics and stimulated depletion (STED) characteristics, the written structure can be subjected to two-photon STED super-resolution microscopic imaging. The present invention utilizes the two-photon and STED effects of the initiator to simultaneously realize super-resolution writing and imaging. Compared with the method of additionally doping fluorescent dyes in the photoresist for super-resolution microscopic imaging, the present invention simplifies the composition of the photoresist. In addition, the same excitation light and loss light can be used in writing and imaging, respectively, so high-precision writing and super-resolution imaging can be realized simultaneously in one optical system. By multiplexing the excitation light and loss light optical paths of writing and imaging, the complexity of the system can be effectively simplified.

[0049] Compared with the prior art, the present invention has the following beneficial technical effects:

[0050] (1) By utilizing the photoinitiator in the photoresist, while achieving two-photon STED high-precision nanostructure engraving, its fluorescence characteristics, especially its two-photon excitation and stimulated emission depletion characteristics, can be used to achieve two-photon STED super-resolution optical microscopy imaging of the engraved structure. There is no need to dope dyes in the photoresist, which simplifies the composition complexity of the photoresist and reduces the impact of doping on the chemical properties of the photoresist.

[0051] (2) The two-photon excitation and loss light used in the imaging process and the two-photon polymerization excitation light and suppression light used in the writing process are in the same optical path, which enables the multiplexing of the writing and imaging excitation light paths, simplifies the system, and makes the system more compact and simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a schematic diagram of the device of the present invention;

[0053] Figure 2 The workflow of the method of the present invention is as follows;

[0054] Figure 3 (a) shows the excitation light (black) and loss light (gray) at the sample surface during lateral super-resolution writing and imaging. The upper figure shows the lateral distribution of the light spot, and the lower figure shows the axial distribution. Figure 3 (b) shows the excitation light (black) and loss light (gray) at the sample surface during axial super-resolution writing and imaging. The upper figure shows the lateral distribution of the light spot, and the lower figure shows the axial distribution.

[0055] FIG4 (a) shows the first working mode based on two-photon STED writing; FIG4 (b) shows the second working mode of writing;

[0056] Figure 5 (a) shows the first working mode based on two-photon STED microscopy; Figure 5 (b) shows the second working mode of writing;

[0057] Figure 6 (a) shows the lateral super-resolution imaging result of ordinary confocal microscopy; Figure 6 (b) shows the lateral super-resolution imaging result of microscopy based on two-photon STED;

[0058] Figure 7 (a) shows the axial super-resolution imaging result of ordinary confocal microscopy; Figure 7 (b) shows the axial super-resolution imaging result of microscopy based on two-photon STED. DETAILED DESCRIPTION

[0059] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings, but the present invention is not limited thereto.

[0060] Embodiment 1

[0061] This embodiment relates to a super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effects, such as Figure 2 As shown, the following steps are included:

[0062] (1) Preparation of photoresist: Add 1% by weight of photoinitiator 7-diethylamino-3-(2-thienyl)coumarin (DETC) to the photoresist monomer;

[0063] (2) Laser direct writing based on two-photon and stimulated emission depletion effects: Two laser beams are focused on the photoresist through an objective lens, one of which is the excitation light, forming a solid spot to induce two-photon polymerization of the photoresist, and the other is the inhibition light, forming a hollow spot to inhibit the polymerization of the photoresist. By aligning the center of the excitation solid light spot with the center of the inhibition hollow light spot, high-precision micro-nano structure super-resolution laser direct writing can be achieved. The writing process can adopt two modes: the excitation light and the inhibition light pass through the objective lens, oil, and substrate, and then converge on the photoresist dripped on the substrate for writing, or pass through the objective lens and converge directly on the photoresist between the objective lens and the substrate for writing;

[0064] (3) Development: Develop the engraved structure and air dry it;

[0065] (4) Two-photon STED super-resolution microscopy based on two-photon and stimulated emission depletion effect: the air-dried inscribed structure is placed back on the sample stage, and two laser beams are focused on the sample through an objective lens. One beam is excitation light, which forms a solid spot to stimulate the initiator to produce fluorescence due to the two-photon effect. The other beam is loss light, which forms a hollow spot. The fluorescence generated at the edge of the loss structure can be realized by aligning the center of the excitation light with the center of the loss light. The imaging process can adopt two modes: the excitation light and the loss light pass through the objective lens, oil, and substrate and converge to the inscribed structure on the upper surface of the substrate for two-photon STED imaging, or pass through the objective lens, oil, and substrate and converge to the inscribed structure immersed on the lower surface of the substrate for two-photon STED imaging;

[0066] Embodiment 2

[0067] like Figure 1 As shown, this embodiment relates to a device for implementing the super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effect of embodiment 1, comprising:

[0068] An excitation light path for initiating two-photon polymerization of photoresist; an inhibition light path for inhibiting polymerization of photoresist; a combined light path of the excitation light path and the inhibition light path for writing; an excitation light path for two-photon excitation of the initiator in the writing structure to produce fluorescence; a loss light path for depleting the initiator to produce fluorescence; a combined light path of the excitation light path and the loss light path for imaging; and a fluorescence imaging detection light path.

[0069] The excitation optical path for inducing two-photon polymerization of the photoresist is sequentially provided with a first femtosecond laser 1 with a wavelength selected between 500-800nm, a pulse pre-compression module 2, a first acousto-optic modulator 3, a first objective lens 4, a first pinhole 5, a first lens 6, and a first half-wave plate 7;

[0070] The inhibition optical path for inhibiting the photoresist from generating two-photon polymerization is sequentially provided with a second laser 8 with a continuous light or ps or ns pulse with a wavelength of 532nm, a second acousto-optic modulator 9, a second objective lens 10, a second pinhole 11, a second lens 12, a first reflector 13, and a second half-wave plate 14;

[0071] The combined beam optical path of the writing excitation light and the inhibition light is sequentially provided with a polarization beam splitter 15, a third half wave plate 16, a spatial light modulator 17, a fourth half wave plate 18, a quarter wave plate 19, a dichroic mirror 20, a scanning galvanometer 21, a scanning lens 22, a second reflecting mirror 23, a field lens 24, a third objective lens 25, a transparent or non-transparent substrate 26, a high-precision piezoelectric three-dimensional displacement stage 27, a sample holder 28, and a photoresist sample 29 containing an initiator. The scanning galvanometer 21, the scanning lens 22, the field lens 24 and the third objective lens 25 form a 4f system at the entrance pupil.

[0072] The excitation light path and loss light path of the imaging are the same as the excitation light path and suppression light path of the writing;

[0073] The combined beam path of the excitation light path and the loss light path for imaging and the combined beam path of the excitation light path and the suppression light path for writing are the same light path;

[0074] The fluorescence imaging detection optical path is sequentially provided with a photoresist sample 29 containing an initiator, a substrate 26, a sample holder 28, a high-precision piezoelectric three-dimensional displacement stage 27, a third objective lens 25, a field lens 24, a second reflector 23, a scanning lens 22, a scanning galvanometer 21, a dichroic mirror 20, a third reflector 30, a filter 31, a third lens 32, a multimode optical fiber 33, and an avalanche diode photodetector 34;

[0075] The computer 35 is also included, which is connected to the first acousto-optic modulator 3 , the second acousto-optic modulator 9 , the spatial light modulator 17 , the scanning galvanometer 21 , the high-precision piezoelectric three-dimensional displacement stage 27 and the avalanche diode photodetector 34 .

[0076] The laser emitted by the first laser is used as the excitation light for inducing the photoresist to produce two-photon polymerization. The excitation light passes through the dispersion pre-compensation module to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The laser after the dispersion compensation passes through the first acousto-optic modulator to control its intensity and switch, and then converges through the first objective lens, and then passes through the first pinhole for spatial filtering to filter out the influence of the preceding optical elements on the quality of the light spot. The first pinhole is located at the focal plane of the first objective lens. The filtered light beam passes through the first lens for collimation and expansion to ensure that the beam diameter after expansion can fill the entrance pupil of the last oil-immersed third objective lens. The expanded laser passes through the first half-wave plate to adjust its linear polarization direction to vertical polarization;

[0077] The laser light emitted by the second laser is used as the inhibiting light for inhibiting the polymerization of the photoresist. The inhibiting light is controlled in intensity and on and off by the second acousto-optic modulator, then converged by the second objective lens, and then spatially filtered by the second pinhole to filter out the influence of the preceding optical element on the quality of the light spot. The second pinhole is located at the focal plane of the second objective lens. The filtered light beam is collimated and expanded by the second lens to ensure that the diameter of the expanded light beam can fill the entrance pupil of the third objective lens that is finally immersed in oil. The expanded inhibiting light is reflected by the first reflector, and then passes through the second half-wave plate to adjust its polarization direction to horizontal polarization.

[0078] The excitation light is reflected by the polarization beam combiner, and the suppression light is transmitted through the polarization beam combiner to realize the combination of the excitation light and the suppression light. The combined light passes through the third half-wave plate and then passes through the spatial light modulator to change the fast and slow axes of the third half-wave plate, and adjust the polarization direction of the suppression light to be consistent with the polarization direction of the spatial light modulator. The spatial light modulator is divided into left and right screens. The left screen loads the corresponding phase to modulate the excitation light into solid light and adjusts the light spot aberration to the minimum. The right screen loads the corresponding phase to modulate the suppression light into hollow light, or cooperates with adjusting the fast and slow axis direction of the third half-wave plate. The left and right screens adjust the light spot in turn, that is, the left screen adjusts the suppression light to be hollow light, and the right screen adjusts the light spot to be hollow light. The screen adjusts the excitation light to be solid light, and the modulated light beam passes through a fourth half-wave plate and a quarter-wave plate, so that the suppression light finally passes through the third objective lens to be a hollow light spot with a dark center to the bottom, and then passes through a dichroic mirror to reflect into a scanning galvanometer to scan the excitation light and the suppression light, and then passes through a scanning lens, and then reflects through a second reflection mirror to enter a field lens, and the field lens collimates the light beam into parallel light and incidents on the third objective lens, and then focuses through the third objective lens into convergent light and incidents on a photoresist configured with an initiator to wait for writing, wherein the excitation light is a solid light spot on the sample surface, and the suppression light is a hollow light with a transverse distribution or an axial distribution that is sleeved on the edge of the excitation light;

[0079] The computer controls the first optical modulator and the second acousto-optic modulator, controls the switching of the laser and the light intensity, and controls the scanning and movement of the scanning galvanometer and the high-precision piezoelectric three-dimensional translation stage to achieve high-precision two-photon STED writing of micro-nano structures.

[0080] The excitation optical path and the loss optical path used in the two-photon STED super-resolution microscopy imaging process are the same optical path as the excitation optical path and the suppression optical path used to achieve high-precision writing;

[0081] The fluorescence emitted by the initiator in the writing structure is received by the third objective lens, then converged by the field lens, reflected by the second reflector, then passed through the scanning lens, and descanned by the scanning galvanometer, then transmitted by the dichroic mirror, then reflected by the third reflector, and then filtered out by the filter to remove stray light caused by laser and environmental factors, and then converged by the third lens. The converged light is then received and transmitted by the multimode optical fiber, and finally the fluorescence signal is detected by the avalanche diode photodetector;

[0082] The computer controls the first acousto-optic modulator and the second acousto-optic modulator to adjust the intensity and switch of the initiator excitation light, controls the scanning galvanometer to scan the light beam, and performs data processing and transmission on the received fluorescence signal to realize two-photon STED super-resolution microscopic imaging of the inscribed structure.

[0083] Embodiment 3

[0084] This embodiment relates to a method for super-resolution writing and imaging using the device of embodiment 2, comprising the following steps:

[0085] (1) The laser light emitted by the first laser 1 is used as the excitation light for inducing two-photon polymerization of the photoresist. The excitation light passes through the pulse pre-compression module 2 to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The laser light after dispersion compensation passes through the first acousto-optic modulator 3 to control its intensity and switch, and then passes through the first objective lens 4 to converge, and then passes through the first pinhole 5 to perform spatial filtering to filter out the influence of the preceding optical elements on the light spot quality. The first pinhole 5 is located at the focal plane of the first objective lens 4. The filtered light beam passes through the first lens 6 to collimate and expand the beam to ensure that the beam diameter after expansion can fill the entrance pupil of the last oil-immersed third objective lens 25. The expanded laser light passes through the first half-wave plate 7 to adjust its linear polarization direction to vertical polarization;

[0086] (2) The laser light emitted by the second laser 8 is used as the inhibiting light for inhibiting the polymerization of the photoresist. The inhibiting light is controlled by the second acousto-optic modulator 9 for intensity and switch, and then converged by the second objective lens 10, and then spatially filtered by the second pinhole 11 to filter out the influence of the preceding optical elements on the quality of the light spot. The second pinhole 11 is located at the focal plane of the second objective lens 10. The filtered light beam is then collimated and expanded by the second lens 12 to ensure that the diameter of the expanded light beam can fill the entrance pupil of the third oil-immersed objective lens 25. The expanded inhibiting light is then reflected by the first reflector 13, and then passes through the second half-wave plate 14 to adjust its polarization direction to horizontal polarization.

[0087] (3) The excitation light is reflected by the polarization beam combiner 15, and the suppression light is transmitted by the polarization beam combiner 15, so as to realize the combination of the excitation light and the suppression light. The combined light passes through the third half-wave plate 16, and then passes through the spatial light modulator 17 to modulate the shape of the light spot, and the fast and slow axes of the third half-wave plate 16 are changed, and the polarization direction of the suppression light is adjusted to be consistent with the polarization direction of the spatial light modulator 17. The spatial light modulator 17 is divided into left and right screens. The left screen is loaded with the corresponding phase to modulate the excitation light into solid light and adjust the light spot aberration to be minimum. The right screen is loaded with the corresponding phase to modulate the suppression light into hollow light. (Or in conjunction with adjusting the fast and slow axis direction of the third half-wave plate 16, the left and right screens adjust the light spot in reverse, that is, the left screen adjusts the suppression light into hollow light, and the right screen adjusts the excitation light into solid light). The modulated light beam passes through the fourth half wave plate 18 and the quarter wave plate 19, so that the suppression light finally passes through the third objective lens 25 to form a hollow light spot with a dark center to the bottom, and then passes through the dichroic mirror 20 to reflect into the scanning galvanometer 21 to scan the excitation light and the suppression light, and then passes through the scanning lens 22, and then reflects through the second reflection mirror 23 to enter the field lens 24, and the field lens 24 collimates the light beam into parallel light and incidents on the third objective lens 25, and then focuses into convergent light through the third objective lens 25 and incidents on the photoresist sample 29 containing the initiator to wait for writing, wherein the excitation light is a solid light spot on the surface of the photoresist sample 29 containing the initiator, and the suppression light is a hollow light with a transverse distribution or an axial distribution that is sleeved on the edge of the excitation light, and the light spot distribution is shown in Figures 3 (a) and 3 (b);

[0088] (4) There are two types of writing modes: the first mode, as shown in FIG4 (a), is to drip oil on the third objective lens 25, and place a substrate 26 (a transparent substrate such as a cover glass or quartz) on the third objective lens 25, and then place the substrate 26 on a sample holder, and the sample holder 28 is placed on a high-precision piezoelectric three-dimensional displacement platform 27, and then drip photoresist on the substrate 26, and the light spot converges on the upper surface of the substrate 26 to write upwards; the second mode, as shown in FIG4 (b), is an immersion writing mode, that is, drip photoresist on the third objective lens 25, and focus the converged light on the lower surface of the substrate 26 (a transparent substrate such as a cover glass or quartz, or a non-transparent substrate such as a silicon wafer), and then write downwards;

[0089] (5) The first AOM 3 and the second AOM 9 are controlled by the computer 35 to realize the switching of the laser and the light intensity control, and the scanning galvanometer 21 and the scanning and movement of the high-precision piezoelectric three-dimensional translation stage 27 are controlled by the computer 35 to realize the two-photon STED writing of high-precision micro-nano structures;

[0090] (6) The engraved micro-nanostructure is developed and then placed back on the high-precision piezoelectric three-dimensional translation stage 27 to wait for two-photon STED super-resolution microscopy imaging of the engraved structure;

[0091] (7) The excitation optical path and the loss optical path used in the two-photon STED super-resolution microscopy imaging process are the same optical path as the excitation optical path and the suppression optical path used to achieve high-precision writing;

[0092] (8) The process of performing two-photon STED super-resolution microscopy imaging also includes two modes: the first mode is shown in FIG. 5 (a), in which the excitation light and the loss light focused by the third objective lens 25 pass through the oil above the third objective lens 25, and then pass through the substrate 26 (a transparent substrate such as a cover glass) to converge on the writing structure on the upper surface of the substrate 26 dripped with oil to achieve two-photon excitation and STED loss of the initiator in the writing structure; the second mode is shown in FIG. 5 (b), in which oil is dripped above the third objective lens 25, and the substrate 26 (lens or non-lens substrate) with the structure engraved is placed downward above the third objective lens 25, and the excitation light and the loss light focused by the third objective lens 25 converge on the writing structure on the lower surface of the substrate to achieve two-photon excitation and STED loss;

[0093] (9) The fluorescence emitted by the initiator in the writing structure is received by the third objective lens 25, then converged by the field lens 24, reflected by the second reflector 23, then passed through the scanning lens 22, and scanned by the scanning galvanometer 21, then transmitted by the dichroic mirror 20, then reflected by the third reflector 30, and then filtered out by the filter 31 to remove the stray light caused by laser and environmental factors, and then converged by the third lens 32. The converged light is then received and transmitted by the multimode optical fiber 33, and finally the fluorescence signal is detected by the avalanche diode photodetector 34;

[0094] (10) The computer 35 outputs control signals to control the first AOM 3 and the second AOM 9 to adjust the intensity of the initiator excitation light and switch, control the scanning galvanometer 21 to scan the light beam, and perform data processing and transmission on the received fluorescence signal to achieve two-photon STED super-resolution microscopy imaging of the inscribed structure. The imaging results are shown in FIG6(a), FIG6(b) and FIG7(a), FIG7(b).

[0095] The above description is only an example of a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effects, comprising the following steps: (1) Preparation of photoresist: adding photoinitiator to photoresist monomer; (2) Laser direct writing based on two-photon and stimulated emission depletion effects: Two laser beams are focused on the photoresist through an objective lens. One beam is the excitation light, which forms a solid spot to induce two-photon polymerization of the photoresist. The other beam is the inhibition light, which forms a hollow spot to inhibit the polymerization of the photoresist. By aligning the center of the excitation solid light spot with the center of the inhibition hollow light spot, super-resolution laser direct writing of high-precision micro-nano structures can be achieved. (3) Development: Develop the engraved structure and air dry it; (4) Two-photon STED super-resolution microscopy based on two-photon and stimulated emission depletion effects: The air-dried inscribed structure is placed back on the sample stage, and two laser beams are focused on the sample through an objective lens. One beam is the excitation light, which forms a solid spot to stimulate the initiator to produce a two-photon effect and emit fluorescence. The other beam is the loss light, which forms a hollow spot. The fluorescence generated at the edge of the loss structure is overlapped by the excitation light and the loss light center to achieve two-photon STED super-resolution microscopy of the inscribed structure.

2. The super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effect as claimed in claim 1, characterized in that: The writing process adopts a mode: the excitation light and the suppression light pass through the objective lens, oil, and substrate, and then converge on the photoresist dropped on the substrate for writing, or pass through the objective lens and converge directly on the photoresist between the objective lens and the substrate for writing.

3. The super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effect as claimed in claim 1, characterized in that: The imaging process adopts a mode: the excitation light and the loss light pass through the objective lens, oil, and substrate, and converge to the inscribed structure on the upper surface of the substrate for two-photon STED imaging, or pass through the objective lens, oil, and substrate, and converge to the inscribed structure immersed in the lower surface of the substrate for two-photon STED imaging.

4. The super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effect as claimed in claim 1, characterized in that: The initiator was 7-diethylamino-3-(2-thienyl)coumarin DETC.

5. The super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effect as claimed in claim 1, characterized in that: The ratio of DETC to the total weight of the mixture of the photoresist material monomer and DETC is 1%.

6. A device for implementing the super-resolution laser direct writing and imaging method based on two-photon and stimulated emission depletion effect as claimed in claim 1, comprising: An excitation light path for inducing two-photon polymerization of the photoresist; An inhibition optical path for inhibiting photoresist polymerization; a combined optical path of an excitation optical path for writing and an inhibition optical path; an excitation optical path for two-photon excitation of an initiator in an engraved structure to generate fluorescence; a loss optical path for depleting an initiator to generate fluorescence; a combined optical path of an excitation optical path for imaging and a loss optical path; and a fluorescence imaging detection optical path; The excitation optical path for inducing two-photon polymerization of the photoresist is provided with a first laser (1), a pulse pre-compression module (2), a first acousto-optic modulator (3), a first objective lens (4), a first pinhole (5), a first lens (6), and a first half-wave plate (7) in sequence; The suppression optical path for suppressing two-photon polymerization of the photoresist is provided with a second laser (8), a second acousto-optic modulator (9), a second objective lens (10), a second pinhole (11), a second lens (12), a first reflector (13), and a second half-wave plate (14) in sequence; The combined beam path of the writing excitation light and the suppression light is provided with a polarization beam splitter (15), a third half-wave plate (16), a spatial light modulator (17), a fourth half-wave plate (18), a quarter-wave plate (19), a dichroic mirror (20), a scanning galvanometer (21), a scanning lens (22), a second reflector (23), a field lens (24), a third objective lens (25), a transparent or non-transparent substrate (26), a high-precision piezoelectric three-dimensional displacement stage (27), a sample holder (28), and a photoresist sample containing an initiator (29); the scanning galvanometer (21), the scanning lens (22), the field lens (24), and the third objective lens (25) form a 4f system at the entrance pupil; The excitation light path and loss light path of the imaging are the same as the excitation light path and suppression light path of the writing; The combined beam path of the excitation light path and the loss light path for imaging and the combined beam path of the excitation light path and the suppression light path for writing are the same light path; The fluorescence imaging detection optical path is provided with a photoresist sample (29) containing an initiator, a substrate (26), a sample holder (28), a high-precision piezoelectric three-dimensional displacement stage (27), a third objective lens (25), a field lens (24), a second reflector (23), a scanning lens (22), a scanning galvanometer (21), a dichroic mirror (20), a third reflector (30), a filter (31), a third lens (32), a multimode optical fiber (33), and an avalanche diode photodetector (34) in sequence; It also includes a computer (35) connected to the first acousto-optic modulator (3), the second acousto-optic modulator (9), the spatial light modulator (17), the scanning galvanometer (21), the high-precision piezoelectric three-dimensional displacement stage (27), and the avalanche diode photodetector (34); The laser emitted by the first laser is used as the excitation light for inducing the photoresist to produce two-photon polymerization. The excitation light passes through the dispersion pre-compensation module to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The laser after the dispersion compensation passes through the first acousto-optic modulator to control its intensity and switch, and then converges through the first objective lens, and then passes through the first pinhole for spatial filtering to filter out the influence of the preceding optical elements on the quality of the light spot. The first pinhole is located at the focal plane of the first objective lens. The filtered light beam passes through the first lens for collimation and expansion to ensure that the beam diameter after expansion can fill the entrance pupil of the last oil-immersed third objective lens. The expanded laser passes through the first half-wave plate to adjust its linear polarization direction to vertical polarization; The laser light emitted by the second laser is used as the inhibiting light for inhibiting the polymerization of the photoresist. The inhibiting light is controlled in intensity and on and off by the second acousto-optic modulator, then converged by the second objective lens, and then spatially filtered by the second pinhole to filter out the influence of the preceding optical element on the quality of the light spot. The second pinhole is located at the focal plane of the second objective lens. The filtered light beam is collimated and expanded by the second lens to ensure that the diameter of the expanded light beam can fill the entrance pupil of the third objective lens that is finally immersed in oil. The expanded inhibiting light is reflected by the first reflector, and then passes through the second half-wave plate to adjust its polarization direction to horizontal polarization. The excitation light is reflected by the polarization beam combiner, and the suppression light is transmitted through the polarization beam combiner to realize the combination of the excitation light and the suppression light. The combined light passes through the third half-wave plate and then passes through the spatial light modulator to change the fast and slow axes of the third half-wave plate, and adjust the polarization direction of the suppression light to be consistent with the polarization direction of the spatial light modulator. The spatial light modulator is divided into left and right screens. The left screen loads the corresponding phase to modulate the excitation light into solid light and adjusts the light spot aberration to the minimum. The right screen loads the corresponding phase to modulate the suppression light into hollow light, or cooperates with adjusting the fast and slow axis direction of the third half-wave plate. The left and right screens adjust the light spot in turn, that is, the left screen adjusts the suppression light to be hollow light, and the right screen adjusts the light spot to be hollow light. The screen adjusts the excitation light to be solid light, and the modulated light beam passes through a fourth half-wave plate and a quarter-wave plate, so that the suppression light finally passes through the third objective lens to be a hollow light spot with a dark center to the bottom, and then passes through a dichroic mirror to reflect into a scanning galvanometer to scan the excitation light and the suppression light, and then passes through a scanning lens, and then reflects through a second reflection mirror to enter a field lens, and the field lens collimates the light beam into parallel light and incidents on the third objective lens, and then focuses through the third objective lens into convergent light and incidents on a photoresist configured with an initiator to wait for writing, wherein the excitation light is a solid light spot on the sample surface, and the suppression light is a hollow light with a transverse distribution or an axial distribution that is sleeved on the edge of the excitation light; The computer controls the first optical modulator and the second acousto-optic modulator, controls the switching and light intensity of the laser, and controls the scanning galvanometer and the scanning and movement of the high-precision piezoelectric three-dimensional translation stage to achieve high-precision two-photon STED writing of micro-nano structures; The computer controls the first optical modulator and the second acousto-optic modulator, controls the switching and light intensity of the laser, and controls the scanning galvanometer and the scanning and movement of the high-precision piezoelectric three-dimensional translation stage to achieve high-precision two-photon STED writing of micro-nano structures; The excitation optical path and the loss optical path used in the two-photon STED super-resolution microscopy imaging process are the same optical path as the excitation optical path and the suppression optical path used to achieve high-precision writing; The fluorescence emitted by the initiator in the writing structure is received by the third objective lens, then converged by the field lens, reflected by the second reflector, then passed through the scanning lens, and descanned by the scanning galvanometer, then transmitted by the dichroic mirror, then reflected by the third reflector, and then filtered out by the filter to remove the stray light caused by the laser and environmental factors, and then converged by the third lens. The converged light is then received and transmitted by the multimode optical fiber, and finally the fluorescence signal is detected by the avalanche diode photodetector; The computer controls the first acousto-optic modulator and the second acousto-optic modulator to adjust the intensity and switch of the initiator excitation light, controls the scanning galvanometer to scan the light beam, and performs data processing and transmission on the received fluorescence signal to realize two-photon STED super-resolution microscopic imaging of the inscribed structure.

7. The device according to claim 6, characterized in that: The first laser (1) is a femtosecond laser with a wavelength between 500nm and 800nm; The second laser (8) is a continuous light laser, or a ps or ns laser, with a wavelength of 532 nm; The diameter of the multimode optical fiber (33) is selected to be the size of one Airy disk diameter after system amplification.

8. The device according to claim 6, characterized in that: The scanning galvanometer (21), the scanning lens (22), the field lens (24) and the entrance pupil of the third objective lens (25) form a 4f system.

9. The device according to claim 6, characterized in that: The substrate (26) is a transparent material such as a cover glass, quartz or sapphire, or a non-transparent material such as a silicon wafer.

10. A method for super-resolution laser direct writing and imaging using the device of any one of claims 6 to 9, characterized in that: The steps include: (1) The laser light emitted by the first laser (1) is used as the excitation light for inducing the photoresist to generate two-photon polymerization. The excitation light passes through the pulse pre-compression module (2) to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The laser light after the dispersion compensation passes through the first acousto-optic modulator (3) to control its intensity and switch, and then passes through the first objective lens (4) to converge, and then passes through the first pinhole (5) to perform spatial filtering to filter out the influence of the preceding optical elements on the light spot quality. The first pinhole (5) is located at the focal plane of the first objective lens (4). The filtered light beam passes through the first lens (6) to collimate and expand the beam to ensure that the diameter of the beam after expansion can fill the entrance pupil of the last oil-immersed third objective lens (25). The expanded laser light passes through the first half-wave plate (7) to adjust its linear polarization direction to vertical polarization; (2) The laser light emitted by the second laser (8) is used as the inhibiting light for inhibiting the polymerization of the photoresist. The inhibiting light is controlled in intensity and on and off by the second acousto-optic modulator (9), then converged by the second objective lens (10), and then spatially filtered by the second pinhole (11) to filter out the influence of the preceding optical element on the quality of the light spot. The second pinhole (11) is located at the focal plane of the second objective lens (10). The filtered light beam is collimated and expanded by the second lens (12) to ensure that the diameter of the expanded light beam can fill the entrance pupil of the third oil-immersed objective lens (25). The expanded inhibiting light is reflected by the first reflector (13), and then passes through the second half-wave plate (14) to adjust its polarization direction to horizontal polarization. (3) The excitation light is reflected by the polarization beam combiner (15), and the suppression light is transmitted by the polarization beam combiner (15), so as to realize the combination of the excitation light and the suppression light. The combined light passes through the third half-wave plate (16), and then passes through the spatial light modulator (17) to modulate the shape of the light spot, and the fast and slow axes of the third half-wave plate (16) are changed, and the polarization direction of the suppression light is adjusted to be consistent with the polarization direction of the spatial light modulator (17). The spatial light modulator (17) is divided into left and right screens. The left screen is loaded with a corresponding phase to modulate the excitation light into solid light and adjust the light spot aberration to be minimum. The right screen is loaded with a corresponding phase to modulate the suppression light into hollow light. Alternatively, the left and right screens adjust the light spot in reverse by adjusting the fast and slow axis directions of the third half-wave plate (16). That is, the left screen adjusts the suppression light into hollow light, and the right screen adjusts the excitation light into solid light. The modulated light beam passes through a fourth half-wave plate (18) and a quarter-wave plate (19), so that the suppression light finally passes through the third objective lens (25) to form a hollow light spot with a dark center and a bottom, and then passes through a dichroic mirror (20) to reflect into a scanning galvanometer (21) to scan the excitation light and the suppression light, and then passes through a scanning lens (22), and then is reflected by a second reflection mirror (23) to enter a field lens (24), wherein the field lens (24) collimates the light beam into parallel light and is incident on the third objective lens (25), and then is focused into convergent light by the third objective lens (25) and is incident on a photoresist sample (29) containing an initiator to wait for writing, wherein the excitation light is a solid light spot on the surface of the photoresist sample (29) containing the initiator, and the suppression light is a hollow light that is laterally distributed or axially distributed and is sheathed on the edge of the excitation light; (4) The writing modes are divided into two types: the first type is to drip oil on the top of the third objective lens (25), and place a substrate (26) on the top of the third objective lens (25), and then place the substrate (26) on a sample holder, and the sample holder (28) is placed on a high-precision piezoelectric three-dimensional displacement stage (27), and then drip photoresist on the substrate (26), and the light spot converges on the upper surface of the substrate (26) to write upwards; the second type is an immersion writing mode, that is, drip photoresist on the top of the third objective lens (25), and focus the converged light on the lower surface of the substrate (26) to write downwards; (5) controlling the first acousto-optic modulator (3) and the second acousto-optic modulator (9) by means of a computer (35) to realize switching of the laser and light intensity control, and controlling the scanning galvanometer (21) and the scanning and movement of the high-precision piezoelectric three-dimensional displacement stage (27) by means of the computer (35) to realize two-photon STED writing of high-precision micro-nano structures; (6) The engraved micro-nanostructure is developed and then placed back on the high-precision piezoelectric three-dimensional translation stage (27) to wait for two-photon STED super-resolution microscopy imaging of the engraved structure; (7) The excitation optical path and the loss optical path used in the two-photon STED super-resolution microscopy imaging process are the same optical path as the excitation optical path and the suppression optical path used to achieve high-precision writing; (8) The process of performing two-photon STED super-resolution microscopy imaging also includes two modes: the first mode is that the excitation light focused by the third objective lens (25) and the loss light pass through the oil above the third objective lens (25), and then pass through the substrate (26) to converge on the writing structure on the upper surface of the substrate (26) dripped with oil to achieve two-photon excitation and STED loss of the initiator in the writing structure; the second mode is to drip oil above the third objective lens (25), and place the substrate (26) with the structure facing downward above the third objective lens (25), and the excitation light and loss light focused by the third objective lens (25) converge on the writing structure on the lower surface of the substrate to achieve two-photon excitation and STED loss; (9) The fluorescence emitted by the initiator in the writing structure is received by the third objective lens (25), then converged by the field lens (24), reflected by the second reflector (23), then passed through the scanning lens (22), and scanned by the scanning galvanometer (21), then transmitted by the dichroic mirror (20), then reflected by the third reflector (30), filtered out by the filter (31) to remove stray light caused by laser and environmental factors, and then converged by the third lens (32). The converged light is then received and transmitted by the multimode optical fiber (33), and finally the fluorescence signal is detected by the avalanche diode photodetector (34); (10) The computer (35) outputs a control signal to control the first acousto-optic modulator (3) and the second acousto-optic modulator (9) to adjust the intensity of the initiator excitation light and switch, control the scanning galvanometer (21) to scan the light beam, and perform data processing and transmission on the received fluorescence signal, thereby realizing two-photon STED super-resolution microscopic imaging of the inscribed structure.

Images