An imaging method and device based on two-photon absorption effect

Abstract

The present invention discloses an imaging method and device based on the two-photon absorption effect, and the method comprises: (1) introducing a photosensitive substance with two-photon absorption characteristics into a sample; (2) using a femtosecond laser to precisely focus the sample, and inducing the formation of a micro-nano structure through two-photon absorption; (3) washing the sample, removing the unreacted photosensitive substance, and air-drying the sample; (4) placing the treated sample back on the sample stage, and using a femtosecond laser to focus on the sample again, stimulating two-photon luminescence, receiving fluorescence and converging it to a detector, and realizing super-resolution microscopic imaging of the micro-nano structure. The present invention utilizes the two-photon polymerization characteristics and fluorescence luminescence characteristics of the photosensitive substance to realize the formation of the micro-nano structure and image it, without the need to additionally dope the sample with dyes, thereby simplifying the material composition of the sample. In addition, the present invention can use the same femtosecond laser to simultaneously realize super-resolution structure construction and imaging, effectively simplifying the system.

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 an imaging method and device based on two-photon absorption effect. Background Art

[0002] Laser direct writing technology is a technology that uses converged lasers to process directly on photoresists. Compared with traditional micro-nano processing technologies such as photolithography, nanoimprinting and electron beam exposure, laser direct writing has the advantages of high flexibility, low cost and low requirements for processing environment in micro-nano processing, making it widely used in many fields, including the manufacture of various micro-machines, microfluidics, micro-optics, microelectronic devices and biomedicine. Traditional laser direct writing technology based on single-photon absorption effect can achieve 2D or 2.5D processing, but cannot truly achieve 3D micro-nano structure engraving. The invention of femtosecond laser has enabled the discovery of the two-photon effect of materials, and has achieved a higher writing resolution in micro-nano processing, achieving high-precision engraving of sub-100 nanometer feature size structures, and at the same time has the flexibility to achieve the ability of three-dimensional micro-nano processing of arbitrary structures.

[0003] The traditional imaging characterization method usually involves developing the written structure, spraying gold, and then observing it with an electron microscope or atomic force microscope. However, electron microscope imaging is slow, has high environmental requirements, and is expensive. In the fields of biomedicine and the like, dyes are usually used to fluorescently label biological cells for super-resolution microscopic imaging; similarly, in order to image the written structure, dyes can be doped into the photoresist, and the fluorescent dyes in the written structure are subsequently imaged. However, the introduction of dyes increases the complexity of the photoresist composition, causing certain changes in its chemical properties, which has a certain impact on the writing process. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide an imaging method and device based on the two-photon absorption effect.

[0005] In laser direct writing, some initiators have the function of initiating photoresist polymerization by two-photon absorption, and thus can be used in two-photon laser direct writing. In addition to the characteristic of initiating polymerization by two-photon absorption in writing, the initiator also has the characteristic of fluorescence emission. In addition to single-photon excitation to produce fluorescence, it can also be subjected to two-photon excitation to produce fluorescence. This characteristic can be used to perform super-resolution optical microscopic imaging characterization on the written structure. The present invention utilizes the two-photon absorption effect of the initiator's luminescence to perform two-photon excitation on the written structure to achieve optical microscopic imaging characterization, which has the advantages of being non-invasive, fast imaging speed, simple operation, and low environmental requirements.

[0006] By utilizing the two-photon absorption effect of the initiator in the photoresist, two functions can be achieved simultaneously: first, the photoresist is triggered to produce two-photon polymerization, thereby realizing laser direct writing of high-precision micro-nano structures; at the same time, there is no need to additionally dope the photoresist with dyes, but the fluorescence luminescence characteristics of the initiator itself are utilized to perform two-photon excitation on it to produce fluorescence and perform imaging, thereby realizing super-resolution microscopic imaging characterization of the written structure. The light beam used by the present invention to trigger the photoresist to produce two-photon polymerization and to perform two-photon excitation on the initiator to produce fluorescence can be the same femtosecond light beam, and by multiplexing the excitation light paths for writing and imaging, the complexity of the device can be effectively simplified.

[0007] The imaging method based on the two-photon absorption effect of the present invention comprises the following steps:

[0008] (1) Preparation of photoresist: adding photoinitiator to photoresist monomer;

[0009] (2) Laser direct writing based on the two-photon absorption effect: A beam of femtosecond laser is focused on the photoresist through an objective lens, inducing two-photon polymerization of the photoresist, thus achieving laser direct writing of high-precision micro-nano structures;

[0010] (3) Development: Develop the engraved structure and air dry it;

[0011] (4) Two-photon microscopy based on two-photon absorption effect: The air-dried inscribed structure is placed back on the sample stage, and a beam of femtosecond laser is focused on the sample through an objective lens to generate two-photon excitation. The excited fluorescence is received by the objective lens and then converged onto a photodetector through a lens for two-photon imaging, thereby achieving two-photon super-resolution microscopy of the inscribed structure.

[0012] Preferably, the writing process can adopt two modes: the femtosecond laser passes through the objective lens, oil, and substrate, and then converges on the photoresist dripped on the substrate for writing, or passes through the objective lens and directly converges on the photoresist between the upper part of the objective lens and the substrate for writing.

[0013] Preferably, the imaging process can adopt two modes: the femtosecond laser passes through the objective lens, oil, and substrate and converges to the inscribed structure on the upper surface of the substrate for two-photon excitation imaging, or passes through the objective lens, oil, and substrate and converges to the inscribed structure immersed in the lower surface of the substrate for two-photon excitation imaging.

[0014] Preferably, the initiator is 7-diethylamino-3-(2-thienyl)coumarin DETC.

[0015] Preferably, the ratio of DETC to the total weight of the mixture of the photoresist material monomer and DETC is 1%.

[0016] The device provided by the present invention comprises the following parts:

[0017] An excitation light path for initiating two-photon polymerization of photoresist; an excitation light path for two-photon excitation of the initiator in the writing structure to generate fluorescence; and a fluorescence imaging detection light path;

[0018] The excitation optical path for inducing the photoresist to produce two-photon polymerization is sequentially provided with a laser, an optical isolator, a pulse pre-compression module, an acousto-optic modulator, a first objective lens, a pinhole, a first lens, a dichroic mirror, a scanning galvanometer, a scanning lens, a first reflector, a field lens, a second objective lens, a high-precision piezoelectric displacement stage, a sample holder, a substrate, and a photoresist sample containing an initiator;

[0019] The fluorescence excitation light path of the initiator and the photoresist polymerization excitation light path are the same light path;

[0020] The fluorescence imaging detection optical path is provided with a photoresist sample containing an initiator, a substrate, a sample holder, a high-precision piezoelectric displacement stage, a second objective lens, a field lens, a first reflector, a scanning lens, a scanning galvanometer, a dichroic mirror, a second reflector, a filter, a second lens, a multimode optical fiber, and an avalanche diode photodetector in sequence;

[0021] It also includes a computer connected to an acousto-optic modulator, a scanning galvanometer, a high-precision piezoelectric translation stage, and an avalanche diode photodetector.

[0022] Preferably, the laser is a femtosecond laser, which is used to excite the photoresist to produce two-photon polymerization, and is also used for two-photon excitation of the initiator to produce fluorescence;

[0023] Preferably, the wavelength of the laser is between 500nm-800nm;

[0024] Preferably, the dichroic mirror is a long-pass dichroic mirror;

[0025] Preferably, the diameter of the multimode optical fiber is selected to be the size of one Airy disk diameter after system amplification;

[0026] Preferably, the scanning galvanometer, the scanning lens, the field lens and the entrance pupil of the second objective lens form a 4f system;

[0027] 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.

[0028] The working process of applying the imaging device based on the two-photon absorption effect of the present invention to perform super-resolution writing and imaging specifically includes the following steps:

[0029] (1) The laser emitted by the femtosecond laser is used as the excitation light for inducing two-photon polymerization of the photoresist. The excitation light passes through an optical isolator to prevent the laser from returning to the laser. The laser then passes through a pulse pre-compression module to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse caused by subsequent optical elements. The laser after dispersion compensation passes through an acousto-optic modulator to control its intensity and switch, and then passes through a first objective lens for convergence, and then passes through a pinhole for spatial filtering to filter out the influence of the preceding optical elements on the light spot quality. The pinhole is located at the focal plane of the first objective lens. The light beam after the wave is collimated and expanded by the first lens to ensure that the diameter of the light beam after expansion can fill the entrance pupil of the second objective lens immersed in oil, and then reflected by the dichroic mirror into the scanning galvanometer to scan the light beam, and the scanned light beam is converged by the scanning lens, and the scanning galvanometer is located at the front focal plane of the scanning lens, so that it generates a scanning fixed point after passing through the scanning lens, and then reflected by the first reflecting mirror into the field lens, and the field lens collimates the light beam into parallel light and incidents on the second objective lens, and then focuses into convergent light through the second objective lens and incidents on the photoresist sample equipped with the initiator to wait for writing;

[0030] (2) There are two modes of writing. The first mode is to drip oil on the second objective lens, and place a substrate (transparent substrate such as cover glass, quartz, etc.) on the second objective lens, and then place the substrate on a sample holder, and the sample holder is placed on a high-precision piezoelectric displacement stage. A photoresist sample containing an initiator is dripped on the substrate, and the light spot is focused on the upper surface of the substrate to write upwards. The second mode is an immersion writing mode, that is, dripping photoresist on the second objective lens, focusing the converged light on the lower surface of the substrate (transparent substrate such as cover glass, quartz, or non-transparent substrate such as silicon wafer), and writing downwards.

[0031] (3) The computer controls the acousto-optic modulator to realize the switching of the laser and the light intensity control, and the computer controls the scanning galvanometer and the scanning and movement of the high-precision piezoelectric displacement stage to realize the two-photon writing of high-precision micro-nano structures;

[0032] (4) The engraved micro-nanostructure is developed and then placed back on the high-precision piezoelectric displacement stage to wait for two-photon super-resolution microscopy imaging;

[0033] (5) The excitation optical path used in the two-photon super-resolution microscopy imaging process is the same as the optical path used to achieve high-precision writing;

[0034] (6) The process of super-resolution microscopic imaging also includes two modes: the first mode is that the convergent light beam focused by the second objective lens passes through the oil above the second objective lens, and then passes through the substrate (transparent substrate such as cover glass) to converge on the inscribed structure on the upper surface of the substrate dripped with oil to achieve two-photon excitation; the second mode is that oil is dripped above the second objective lens, and the substrate with the structure (lens or non-lens substrate) is placed downward above the second objective lens, and the convergent light focused by the second objective lens converges on the inscribed structure on the lower surface of the substrate to achieve two-photon excitation;

[0035] (7) The fluorescence emitted by the initiator in the writing structure is received by the second objective lens, then converged by the field lens, reflected by the first reflector, then passed through the scanning lens, and descanned by the scanning galvanometer, then transmitted through the dichroic mirror, then reflected by the second reflector, and then filtered out by the filter to remove stray light caused by laser and environmental factors, and then converged by the second 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;

[0036] (8) The computer outputs a control signal to control the acousto-optic modulator to adjust the intensity of the initiator excitation light and switch it on and off, and controls the scanning galvanometer to scan the light beam, and performs data processing and transmission on the received fluorescence signal to achieve two-photon super-resolution microscopic imaging of the inscribed structure.

[0037] The present invention adds a photoinitiator to the photoresist and utilizes its two-photon absorption effect to realize two-photon high-precision laser direct writing; at the same time, by utilizing its own fluorescent luminescence characteristics, especially utilizing its two-photon absorption effect, two-photon super-resolution microscopic imaging can be performed. The present invention utilizes the two-photon absorption polymerization characteristics and the two-photon excitation luminescence characteristics in 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 present invention can use the same beam of excitation light in writing and imaging at the same time, so that high-precision writing and super-resolution imaging can be realized simultaneously in the same optical system, and the system complexity can be effectively simplified by multiplexing the excitation light optical paths of writing and imaging.

[0038] Compared with the prior art, the present invention has the following beneficial technical effects:

[0039] (1) By utilizing the photoinitiator in the photoresist, two-photon high-precision nanostructure writing can be achieved while utilizing its two-photon excited fluorescence characteristics to realize two-photon super-resolution optical microscopy imaging. 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.

[0040] (2) During the engraving process, the two-photon polymerization excitation light used for engraving and the two-photon excitation light used for initiator imaging are the same laser, which realizes the multiplexing of the engraving and imaging excitation light paths, simplifies the system, and makes the system more compact and simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the device of the present invention;

[0042] Figure 2 Schematic diagram of the workflow of the method of the present invention;

[0043] FIG3 (a) is a schematic diagram of a first working mode based on two-photon writing; FIG3 (b) is a schematic diagram of a second working mode of writing;

[0044] FIG4 (a) is a schematic diagram of a first working mode based on two-photon microscopy imaging; FIG4 (b) is a schematic diagram of a second working mode of writing;

[0045] FIG5(a) is a confocal microscopy imaging result based on single-photon excitation; FIG5(b) is a confocal microscopy imaging result based on two-photon excitation. DETAILED DESCRIPTION

[0046] 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.

[0047] Embodiment 1

[0048] This embodiment relates to an imaging method based on two-photon absorption effect of the invention, such as Figure 2 As shown, the following steps are included:

[0049] (1) Preparation of photoresist: Add 1% by weight of photoinitiator 7-diethylamino-3-(2-thienyl)coumarin DETC to the photoresist monomer;

[0050] (2) Laser direct writing based on the two-photon absorption effect: A beam of femtosecond laser is focused on the photoresist through an objective lens, inducing two-photon polymerization of the photoresist, thereby achieving laser direct writing of high-precision micro-nano structures. The writing process can be carried out in two modes: the femtosecond laser passes through the objective lens, oil, and substrate, and then converges on the photoresist dripped on the substrate for writing, or the femtosecond laser passes through the objective lens and directly converges on the photoresist between the upper part of the objective lens and the substrate for writing;

[0051] (3) Development: Develop the engraved structure and air dry it;

[0052] (4) Two-photon microscopic imaging based on two-photon absorption effect: the air-dried inscribed structure is placed back on the sample stage, a beam of femtosecond laser is focused on the sample through an objective lens to generate two-photon excitation, the excited fluorescence is received by the objective lens, and then converged to the photodetector through a lens for two-photon imaging, thereby realizing two-photon super-resolution microscopic imaging of the inscribed structure. The imaging process can adopt two modes: the femtosecond laser passes through the objective lens, oil, and substrate, and converges to the inscribed structure on the upper surface of the substrate for two-photon excitation imaging, or passes through the objective lens, oil, and substrate, and converges to the inscribed structure immersed on the lower surface of the substrate for two-photon excitation imaging.

[0053] The device used in the method of this embodiment includes: an excitation light path for initiating two-photon polymerization of photoresist; an excitation light path for two-photon excitation of the initiator in the writing structure to generate fluorescence; and a fluorescence imaging detection light path.

[0054] The excitation optical path for inducing the photoresist to produce two-photon polymerization is sequentially provided with a femtosecond laser 1 with a wavelength of 532nm, an optical isolator 2, a pulse pre-compression module 3, an acousto-optic modulator 4, a first objective lens 5, a pinhole 6, a first lens 7, a long-pass dichroic mirror 8, a scanning galvanometer 9, a scanning lens 10, a first reflector 11, a field lens 12, a second objective lens 13, a transparent or non-transparent substrate 14, a high-precision piezoelectric displacement stage 15, a sample holder 16, and a photoresist sample 17 containing an initiator. The scanning galvanometer 9, the scanning lens 10, the field lens 12, and the entrance pupil of the second objective lens 13 form a 4f system;

[0055] The fluorescence excitation light path of the initiator and the photoresist polymerization excitation light path are the same light path;

[0056] The fluorescence imaging detection optical path is sequentially provided with a photoresist sample 17 containing an initiator, a substrate 14, a sample holder 16, a high-precision piezoelectric displacement stage 15, a second objective lens 13, a field lens 12, a first reflector 11, a scanning lens 10, a scanning galvanometer 9, a dichroic mirror 8, a second reflector 18, a filter 19, a second lens 20, a multimode optical fiber 21 with a diameter of one Airy disk after system amplification, and an avalanche diode photodetector 22;

[0057] The computer 23 is connected to the acousto-optic modulator 4 , the scanning galvanometer 9 , the high-precision piezoelectric displacement stage 15 and the avalanche diode photodetector 22 .

[0058] The working principle of the device is:

[0059] The laser emitted by the femtosecond laser 1 is used as the excitation light for inducing the photoresist to produce two-photon polymerization. The excitation light passes through the optical isolator 2 to prevent the laser from returning to the laser 1. The laser then passes through the pulse pre-compression module 3 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 acousto-optic modulator 4 to control its intensity and switch, and then passes through the first objective lens 5 for convergence, and then passes through the pinhole 6 for spatial filtering to filter out the influence of the preceding optical elements on the spot quality. The pinhole 6 is located at the focal plane of the first objective lens 5. The filtered light beam passes through the optical pre-compression module 3 to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The light beam is collimated and expanded by the first lens 7 to ensure that the diameter of the light beam after expansion can fill the entrance pupil of the second oil-immersed objective lens 13, and then reflected by the dichroic mirror 8 to enter the scanning galvanometer 9 to scan the light beam. The scanned light beam is then converged by the scanning lens 10. The scanning galvanometer 9 is located at the front focal plane of the scanning lens so that it generates a scanning fixed point after passing through the scanning lens. The light beam is then reflected by the first reflecting mirror 11 to enter the field lens 12. The field lens 12 collimates the light beam into parallel light and is incident on the second objective lens 13. The light beam is then focused by the second objective lens 13 into converged light and is incident on the photoresist sample 17 provided with an initiator to wait for writing.

[0060] There are two modes of writing. The first mode is as shown in FIG3 (a), in which oil is dripped on the second objective lens 13, and a substrate 14 (a transparent substrate such as a cover glass or quartz) is placed on the second objective lens 13, and then the substrate 14 is placed on a sample holder 16, and the sample holder is placed on a high-precision piezoelectric displacement stage 15. A photoresist sample 17 containing an initiator is dripped on the substrate 14, and the light spot is converged on the upper surface of the substrate 14 to write upwards. The second mode is as shown in FIG3 (b), which is an immersion writing mode, that is, photoresist is dripped on the second objective lens 13, and the converged light is focused on the lower surface of the substrate 14 (a transparent substrate such as a cover glass or quartz, or a non-transparent substrate such as a silicon wafer), and writing is performed downwards.

[0061] The computer 23 is used to control the acousto-optic modulator 4 to realize the switching of the laser and the light intensity control, and the computer 23 is used to control the scanning galvanometer 9 and the scanning and movement of the high-precision piezoelectric displacement stage 15 to realize the two-photon writing of high-precision micro-nano structures;

[0062] The engraved micro-nanostructure is developed and then placed back on the high-precision three-dimensional piezoelectric displacement stage to wait for two-photon super-resolution microscopy imaging.

[0063] The excitation optical path used in the two-photon super-resolution microscopy imaging process is the same as the optical path used to achieve high-precision writing;

[0064] The process of performing super-resolution two-photon microscopy imaging also includes two modes. The first mode, as shown in FIG4 (a), is that the convergent light beam focused by the second objective lens 13 passes through the oil above the second objective lens 13, and then passes through the substrate 14 (a transparent substrate such as a cover glass) to converge on the inscribed structure on the upper surface of the substrate 14 dripped with oil to achieve two-photon excitation; the second mode, as shown in FIG4 (b), is that oil is dripped above the second objective lens 13, and the substrate 14 (lens or non-lens substrate) with the structure engraved is placed downward above the second objective lens 13, and the convergent light converged by the second objective lens 13 converges on the inscribed structure on the lower surface of the substrate 14 to achieve two-photon excitation;

[0065] The fluorescence emitted by the initiator in the writing structure is received by the second objective lens 13, then converged by the field lens 12, reflected by the first reflector 11, then passed through the scanning lens 10, and descanned by the scanning galvanometer 9, then transmitted by the dichroic mirror 8, then reflected by the second reflector 18, and then filtered out the stray light caused by laser and environmental factors by the filter 19, and then converged by the second lens 20. The converged light is then received and transmitted by the multimode optical fiber 21, and finally the fluorescence signal is detected by the avalanche diode photodetector 22;

[0066] The computer 23 outputs a control signal to control the acousto-optic modulator 4 to adjust the intensity and switch of the initiator excitation light, and controls the scanning galvanometer 9 to scan the light beam, and performs data processing and transmission on the received fluorescence signal to achieve two-photon super-resolution microscopic imaging of the inscribed structure. The two-photon imaging results are shown in Figures 5 (a) and 5 (b). The results show that fluorescence microscopic imaging based on two-photon excitation has higher lateral and axial resolution than that based on single-photon excitation.

[0067] Embodiment 2

[0068] like Figure 1 As shown, this embodiment relates to an imaging device based on two-photon absorption effect of the present invention, comprising:

[0069] An excitation light path for initiating two-photon polymerization of photoresist; an excitation light path for two-photon excitation of the initiator in the engraved structure to cause it to produce fluorescence; and a fluorescence imaging detection light path.

[0070] The excitation optical path for inducing the photoresist to produce two-photon polymerization is sequentially provided with a femtosecond laser 1 with a wavelength of 532nm, an optical isolator 2, a pulse pre-compression module 3, an acousto-optic modulator 4, a first objective lens 5, a pinhole 6, a first lens 7, a long-pass dichroic mirror 8, a scanning galvanometer 9, a scanning lens 10, a first reflector 11, a field lens 12, a second objective lens 13, a transparent or non-transparent substrate 14, a high-precision piezoelectric displacement stage 15, a sample holder 16, and a photoresist sample 17 containing an initiator. The scanning galvanometer 9, the scanning lens 10, the field lens 12, and the entrance pupil of the second objective lens 13 form a 4f system;

[0071] The fluorescence excitation light path of the initiator and the photoresist polymerization excitation light path are the same light path;

[0072] The fluorescence imaging detection optical path is sequentially provided with an initiator photoresist sample 17, a substrate 14, a sample holder 16, a high-precision piezoelectric displacement stage 15, a second objective lens 13, a field lens 12, a first reflector 11, a scanning lens 10, a scanning galvanometer 9, a dichroic mirror 8, a second reflector 18, a filter 19, a second lens 20, a multimode optical fiber 21 with a diameter of one Airy disk after system amplification, and an avalanche diode photodetector 22;

[0073] The computer 23 is connected to the acousto-optic modulator 4 , the scanning galvanometer 9 , the high-precision piezoelectric displacement stage 15 and the avalanche diode photodetector 22 .

[0074] The working principle of the device is:

[0075] The laser emitted by the femtosecond laser 1 is used as the excitation light for inducing the photoresist to produce two-photon polymerization. The excitation light passes through the optical isolator 2 to prevent the laser from returning to the laser 1. The laser then passes through the pulse pre-compression module 3 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 acousto-optic modulator 4 to control its intensity and switch, and then passes through the first objective lens 5 for convergence, and then passes through the pinhole 6 for spatial filtering to filter out the influence of the preceding optical elements on the quality of the light spot. The pinhole 6 is located at the focal plane of the first objective lens 5. The filtered light beam passes through the optical pre-compression module 3 to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse by the subsequent optical elements. The light beam is collimated and expanded by the first lens 7 to ensure that the diameter of the light beam after expansion can fill the entrance pupil of the second oil-immersed objective lens 13, and then reflected by the dichroic mirror 8 to enter the scanning galvanometer 9 to scan the light beam. The scanned light beam is then converged by the scanning lens 10. The scanning galvanometer 9 is located at the front focal plane of the scanning lens so that it generates a scanning fixed point after passing through the scanning lens. The light beam is then reflected by the first reflecting mirror 11 to enter the field lens 12. The field lens 12 collimates the light beam into parallel light and is incident on the second objective lens 13. The light beam is then focused by the second objective lens 13 into converged light and is incident on the photoresist sample 17 provided with an initiator to wait for writing.

[0076] There are two modes of writing. The first mode is as shown in FIG3 (a), in which oil is dripped on the second objective lens 13, and a substrate 14 (a transparent substrate such as a cover glass or quartz) is placed on the second objective lens 13, and then the substrate 14 is placed on a sample holder 16, and the sample holder is placed on a high-precision piezoelectric displacement stage 15. A photoresist sample 17 containing an initiator is dripped on the substrate 14, and the light spot is converged on the upper surface of the substrate 14 to write upwards. The second mode is as shown in FIG3 (b), which is an immersion writing mode, that is, photoresist is dripped on the second objective lens 13, and the converged light is focused on the lower surface of the substrate 14 (a transparent substrate such as a cover glass or quartz, or a non-transparent substrate such as a silicon wafer), and writing is performed downwards.

[0077] The computer 23 is used to control the acousto-optic modulator 4 to realize the switching of the laser and the light intensity control, and the computer 23 is used to control the scanning galvanometer 9 and the scanning and movement of the high-precision piezoelectric displacement stage 15 to realize the two-photon writing of high-precision micro-nano structures;

[0078] The engraved micro-nanostructure is developed and then placed back on the high-precision piezoelectric displacement stage to wait for two-photon super-resolution microscopy imaging.

[0079] The excitation optical path used in the two-photon super-resolution microscopy imaging process is the same as the optical path used to achieve high-precision writing;

[0080] The process of performing super-resolution two-photon microscopy imaging also includes two modes. The first mode, as shown in FIG4 (a), is that the convergent light beam focused by the second objective lens 13 passes through the oil above the second objective lens 13, and then passes through the substrate 14 (a transparent substrate such as a cover glass) to converge on the inscribed structure on the upper surface of the substrate 14 dripped with oil to achieve two-photon excitation; the second mode, as shown in FIG4 (b), is that oil is dripped above the second objective lens 13, and the substrate 14 (lens or non-lens substrate) with the structure engraved is placed downward above the second objective lens 13, and the convergent light converged by the second objective lens 13 converges on the inscribed structure on the lower surface of the substrate 14 to achieve two-photon excitation;

[0081] The fluorescence emitted by the initiator in the writing structure is received by the second objective lens 13, then converged by the field lens 12, reflected by the first reflector 11, then passed through the scanning lens 10, and descanned by the scanning galvanometer 9, then transmitted by the dichroic mirror 8, then reflected by the second reflector 18, and then filtered out the stray light caused by laser and environmental factors by the filter 19, and then converged by the second lens 20. The converged light is then received and transmitted by the multimode optical fiber 21, and finally the fluorescence signal is detected by the avalanche diode photodetector 22;

[0082] The computer 23 outputs a control signal to control the acousto-optic modulator 4 to adjust the intensity and switch of the initiator excitation light, and controls the scanning galvanometer 9 to scan the light beam, and performs data processing and transmission on the received fluorescence signal to achieve two-photon super-resolution microscopic imaging of the inscribed structure. The two-photon imaging results are shown in Figures 5 (a) and 5 (b). The results show that fluorescence microscopic imaging based on two-photon excitation has higher lateral and axial resolution than that based on single-photon excitation.

[0083] Embodiment 3

[0084] This embodiment relates to the working process of the imaging device based on the two-photon absorption effect of the second embodiment:

[0085] (1) The laser light emitted by the femtosecond laser 1 is used as the excitation light for inducing two-photon polymerization of the photoresist. The excitation light passes through the optical isolator 2 to prevent the laser light from returning to the laser 1. The laser light then passes through the pulse pre-compression module 3 to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse caused by the subsequent optical elements. The laser light after dispersion compensation passes through the acousto-optic modulator 4 to control its intensity and switch, and then passes through the first objective lens 5 for convergence, and then passes through the pinhole 6 for spatial filtering to filter out the influence of the preceding optical elements on the light spot quality. The pinhole 6 is located at the focal plane of the first objective lens 5. The filtered light beam is then The light beam is collimated and expanded by the first lens 7 to ensure that the diameter of the light beam after expansion can fill the entrance pupil of the second oil-immersed objective lens 13, and then reflected by the dichroic mirror 8 to enter the scanning galvanometer 9 to scan the light beam. The scanned light beam is then converged by the scanning lens 10. The scanning galvanometer 9 is located at the front focal plane of the scanning lens so that it generates a scanning fixed point after passing through the scanning lens. The light beam is then reflected by the first reflecting mirror 11 to enter the field lens 12. The field lens 12 collimates the light beam into parallel light and incidents on the second objective lens 13. The light beam is then focused by the second objective lens 13 into converged light and incidents on the photoresist sample 17 provided with an initiator to wait for writing.

[0086] (2) There are two modes of writing. The first mode is as shown in FIG3 (a), in which oil is dripped on the second objective lens 13, and a substrate 14 (a transparent substrate such as a cover glass or quartz) is placed on the second objective lens 13. The substrate 14 is then placed on a sample holder 16. The sample holder is placed on a high-precision piezoelectric displacement stage 15. A photoresist sample 17 containing an initiator is dripped on the substrate 14, and the light spot is focused on the upper surface of the substrate 14 to write upward. The second mode is as shown in FIG3 (b), which is an immersion writing mode, that is, photoresist is dripped on the second objective lens 13, and the focused light is focused on the lower surface of the substrate 14 (a transparent substrate such as a cover glass or quartz, or a non-transparent substrate such as a silicon wafer) to write downward.

[0087] (3) The computer 23 is used to control the acousto-optic modulator 4 to realize the switching of the laser and the light intensity control, and the computer 23 is used to control the scanning galvanometer 9 and the scanning and movement of the high-precision piezoelectric displacement stage 15 to realize the two-photon writing of high-precision micro-nano structures;

[0088] (4) The engraved micro-nanostructure is developed and then placed back on the high-precision piezoelectric displacement stage to wait for two-photon super-resolution microscopy imaging;

[0089] (5) The excitation optical path used in the two-photon super-resolution microscopy imaging process is the same as the optical path used to achieve high-precision writing;

[0090] (6) The process of performing super-resolution two-photon microscopy imaging also includes two modes. The first mode, as shown in FIG4 (a), is that the convergent light beam focused by the second objective lens 13 passes through the oil above the second objective lens 13, and then passes through the substrate 14 (a transparent substrate such as a cover glass) to converge on the inscribed structure on the upper surface of the substrate 14 dripped with oil to achieve two-photon excitation; the second mode, as shown in FIG4 (b), is that oil is dripped above the second objective lens 13, and the substrate 14 (lens or non-lens substrate) with the structure engraved is placed downward above the second objective lens 13, and the convergent light focused by the second objective lens 13 converges on the inscribed structure on the lower surface of the substrate 14 to achieve two-photon excitation;

[0091] (7) The fluorescence emitted by the initiator in the writing structure is received by the second objective lens 13, then converged by the field lens 12, reflected by the first reflector 11, then passed through the scanning lens 10, and descanned by the scanning galvanometer 9, then transmitted by the dichroic mirror 8, then reflected by the second reflector 18, and then filtered out by the filter 19 to remove stray light caused by laser and environmental factors, and then converged by the second lens 20. The converged light is then received and transmitted by the multimode optical fiber 21, and finally the fluorescence signal is detected by the avalanche diode photodetector 22;

[0092] (8) The computer 23 outputs a control signal to control the acousto-optic modulator 4 to adjust the intensity of the initiator excitation light and switch it, and controls the scanning galvanometer 9 to scan the light beam, and performs data processing and transmission on the received fluorescence signal to achieve two-photon super-resolution microscopic imaging of the inscribed structure. The two-photon imaging results are shown in Figures 5 (a) and 5 (b). The results show that fluorescence microscopic imaging based on two-photon excitation has higher lateral and axial resolution than that based on single-photon excitation.

[0093] 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. An imaging method based on two-photon absorption effect, comprising the following steps: Step 1, preparation of photoresist: adding photoinitiator to photoresist monomer Step 2, laser direct writing based on two-photon absorption effect: a beam of femtosecond laser is focused on the photoresist through an objective lens, inducing two-photon polymerization of the photoresist, thereby achieving laser direct writing of high-precision micro-nano structures; Step 3, development: developing the engraved structure and air-drying it; Step 4, two-photon microscopy based on two-photon absorption effect: put the air-dried inscribed structure back on the sample stage, focus a beam of femtosecond laser on the sample through an objective lens to produce two-photon excitation, use the objective lens to receive the excited fluorescence, and then converge it onto the photodetector through a lens for two-photon imaging, thereby realizing two-photon super-resolution microscopy of the inscribed structure.

2. The imaging method based on two-photon absorption effect as claimed in claim 1, characterized in that: The laser direct writing process described in step 2 is: the femtosecond laser passes through the objective lens, oil, and substrate, and then converges on the photoresist dropped on the substrate for writing.

3. The imaging method based on two-photon absorption effect as claimed in claim 1, characterized in that: The laser direct writing process described in step 2 is: the laser light passing through the objective lens is directly focused on the photoresist between the upper part of the objective lens and the substrate for writing.

4. The imaging method based on two-photon absorption effect as claimed in claim 1, characterized in that: The two-photon microscopic imaging process described in step 4 is: the femtosecond laser passes through the objective lens, oil, and substrate, and converges to the inscribed structure on the upper surface of the substrate for two-photon excitation imaging.

5. The imaging method based on two-photon absorption effect as claimed in claim 1, characterized in that: The two-photon microscopic imaging process described in step 4 is: through the objective lens and oil, the inscribed structure immersed on the lower surface of the substrate is converged to perform two-photon excitation imaging.

6. The imaging method based on two-photon absorption effect as claimed in claim 1, characterized in that: The photoinitiator in step 1 is 7-diethylamino-3-(2-thienyl)coumarin DETC.

7. The imaging method based on two-photon absorption effect as claimed in claim 6, characterized in that: The ratio of DETC to the total weight of the mixture of the photoresist material monomer and DETC is 1%.

8. A device for implementing the imaging method based on two-photon absorption effect as claimed in claim 1, comprising: An excitation light path for initiating two-photon polymerization of photoresist; an excitation light path for two-photon excitation of the initiator in the writing structure to generate fluorescence; and a fluorescence imaging detection light path; The excitation optical path for inducing two-photon polymerization of the photoresist is provided with a laser (1), an optical isolator (2), a pulse pre-compression module (3), an acousto-optic modulator (4), a first objective lens (5), a pinhole (6), a first lens (7), a dichroic mirror (8), a scanning galvanometer (9), a scanning lens (10), a first reflector (11), a field lens (12), a second objective lens (13), a transparent or non-transparent substrate (14), a high-precision piezoelectric displacement stage (15), a sample holder (16), and a photoresist sample containing an initiator (17); The fluorescence excitation light path of the initiator and the photoresist polymerization excitation light path are the same light path; The fluorescence imaging detection optical path is provided with a photoresist sample (17) containing an initiator, a substrate (14), a sample holder (16), a high-precision piezoelectric displacement stage (15), a second objective lens (13), a field lens (12), a first reflector (11), a scanning lens (10), a scanning galvanometer (9), a dichroic mirror (8), a second reflector (18), a filter (19), a second lens (20), a multimode optical fiber (21), and an avalanche diode photodetector (22) in sequence; The computer (23) is connected to an acousto-optic modulator (4), a scanning galvanometer (9), a high-precision piezoelectric displacement stage (15), and an avalanche diode photodetector (22).

9. The device according to claim 8, characterized in that: The laser (1) is a femtosecond laser, which is used to excite the photoresist to produce two-photon polymerization, and is also used for two-photon excitation of the initiator to produce fluorescence.

10. The device according to claim 8, characterized in that: The wavelength of the laser (1) is between 500nm and 800nm.

11. The device according to claim 8, characterized in that: The dichroic mirror (8) is a long-pass dichroic mirror.

12. The device according to claim 8, characterized in that: The diameter of the multimode optical fiber (21) is selected to be the size of one Airy disk diameter after system amplification.

13. The device according to claim 8, characterized in that: The scanning galvanometer (9), the scanning lens (10), the field lens (12) and the entrance pupil of the second objective lens (13) form a 4f system.

14. The device according to claim 8, characterized in that: The substrate (14) is made of one of the following transparent materials: cover glass, quartz, sapphire, or silicon wafer.

15. A method for imaging using the device according to any one of claims 8 to 14, characterized in that: The steps include: S1. The laser light emitted by the laser (1) is used as the excitation light for inducing the photoresist to generate two-photon polymerization. The excitation light passes through the optical isolator (2) to prevent the laser light from returning to the laser (1). The laser light then passes through the pulse pre-compression module (3) to compress the femtosecond laser pulse to compensate for the dispersion broadening of the femtosecond laser pulse caused by the subsequent optical elements. The laser light after the dispersion compensation passes through the acousto-optic modulator (4) to control its intensity and switch. The laser light then passes through the first objective lens (5) to converge. The laser light then passes through the pinhole (6) to perform spatial filtering to filter out the influence of the preceding optical elements on the light spot quality. The pinhole (6) is located at the focal plane of the first objective lens (5). The filtered light beam then passes through the first lens (5). The mirror (7) is used to collimate and expand the beam, so as to ensure that the diameter of the beam after expansion can fill the entrance pupil of the last oil-immersed second objective lens (13), and then the beam is reflected by the dichroic mirror (8) and enters the scanning galvanometer (9) to scan the beam. The scanned beam is then converged by the scanning lens (10). The scanning galvanometer (9) is located at the front focal plane of the scanning lens so that it generates a scanning fixed point after passing through the scanning lens. The beam is then reflected by the first reflection mirror (11) and enters the field lens (12). The field lens (12) collimates the beam into parallel light and enters the second objective lens (13). The beam is then focused by the second objective lens (13) into converged light and is incident on a photoresist sample (17) containing an initiator to wait for writing. S2. There are two types of writing modes. The first mode is to drip oil on the second objective lens (13), place a substrate (14) on the second objective lens (13), and then place the substrate (14) on a sample holder (16). The sample holder is placed on a high-precision piezoelectric displacement stage (15). A photoresist sample (17) containing an initiator is dripped on the substrate (14), and the light spot is converged on the upper surface of the substrate (14) to write upwards. The second mode is an immersion writing mode, that is, photoresist is dripped on the second objective lens (13), and the converged light is focused on the lower surface of the substrate (14) to write downwards. S3, controlling the acousto-optic modulator (4) through a computer (23) to realize switching of the laser and light intensity control, and controlling the scanning galvanometer (9) and the scanning and movement of the high-precision piezoelectric displacement stage (15) through the computer (23) to realize two-photon writing of high-precision micro-nano structures; S4, completing the development process of the engraved micro-nanostructure, and then placing it back on the high-precision piezoelectric displacement stage to wait for two-photon super-resolution microscopy imaging; S5. The excitation optical path used in the two-photon super-resolution microscopy imaging process is the same as the optical path used to achieve high-precision writing; S6. The process of performing super-resolution microscopic imaging includes two modes: the first mode is that the convergent light beam focused by the second objective lens (13) passes through the oil above the second objective lens (13), and then passes through the substrate (14), and converges to the inscribed structure on the upper surface of the substrate (14) dripped with oil to achieve two-photon excitation; the second mode is that oil is dripped above the second objective lens (13), and the substrate (14) with the structure engraved is placed downward above the second objective lens (13), and the convergent light focused by the second objective lens (13) is converged on the inscribed structure on the lower surface of the substrate (14) to achieve two-photon excitation; S7, the fluorescence emitted by the initiator in the inscribed structure is received by the second objective lens (13), then converged by the field lens (12), reflected by the first reflector (11), then passed through the scanning lens (10), and scanned by the scanning galvanometer (9), then transmitted by the dichroic mirror (8), then reflected by the second reflector (18), then filtered out by the filter (19) to remove stray light caused by laser and environmental factors, and then converged by the second lens (20), the converged light is then received and transmitted by the multimode optical fiber (21), and finally the fluorescence signal is detected by the avalanche diode photodetector (22); S8. The computer (23) outputs a control signal to control the acousto-optic modulator (4) to adjust the intensity of the initiator excitation light and switch it on and off, and controls the scanning galvanometer (9) to scan the light beam, and performs data processing and transmission on the received fluorescence signal, thereby realizing two-photon super-resolution microscopic imaging of the inscribed structure.

Images