CN103712965A - Resolution testing standard board manufacturing method for super-resolution fluorescence microscope system - Google Patents
Resolution testing standard board manufacturing method for super-resolution fluorescence microscope system Download PDFInfo
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Abstract
The invention discloses a resolution testing standard board manufacturing method for a super-resolution fluorescence microscope system. A nano-manufacturing technology and a nanoparticle self-assembling technology are used for manufacturing a fluorescent quantum dot line with a width of 20 to 100nm as a fluorescent super-resolution standard board. At present, a main means for detecting the resolution of a super-resolution fluorescence microscope is fluorescent nanospheres, but is very complex. The manufacturing method is simple and reliable, excellent fluorescent characteristics are ensured, and the manufactured super-resolution standard board can be directly used for testing the super-resolution fluorescence microscope system.
Description
Technical field
The present invention relates to the resolution detection technique field of super-resolution fluorescence microscopic system, be specially a kind of manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate.
Background technology
In fields such as biomedicines, for understanding better the mechanism of life and the mechanism of production of disease, need accurate location and the distribution of the fine structures such as the interior organ of observation of cell, illustrate the basic structure how biomacromolecules such as albumen form cell, how important active factors regulates the main vital movement of cell etc.
The characteristic dimension of above-mentioned system is in nanometer scale, and conventional optical microscopy cannot meet observation requirements.For further improving the resolution of optical microscope, be mainly to discuss based on diffraction limit formula in early days, by reducing operation wavelength and increasing the size that numerical aperture is compressed focal beam spot.The technology such as scanning electron microscope that grow up based on reducing operation wavelength, though can realize nano level resolution, larger to sample destructiveness, be applicable to surface observation, and be not suitable for biological sample, the particularly observation of live body sample.Along with the appearance of Novel fluorescent molecular probe and the development of imaging technique, super-resolution fluorescence microtechnic is arisen at the historic moment.At present, the super-resolution microtechnic of main flow mainly comprises two classes, one class is random switching and reads microtechnic, utilize unimolecule localization method to realize super-resolution imaging, comprise the super-resolution microtechnics such as photosensitive location microscopy (PALM), the photosensitive location of fluorescence microscopy (FPALM) and random optics reconstruction microscopy (STORM).There is certain defect in this class technology: in observation process, needs reconditioning-quench fluorescence molecule, cause observation speed slower, be not suitable for and observe live body sample.Another kind of is target switch and read microtechnic, comprises the super-resolution microscopies such as stimulated emission loss microscopy (STED), Structured Illumination microscopy (SIM) and ground state loss microscopy (GSD).Studying many is STED, has mainly utilized the nonlinear effect of fluorescence to reduce the size of focal beam spot, at aspects such as dynamic image acquisition capability, theoretical resolution limitation and three-dimensional super-resolution imagings, has the advantage that other similar approach hardly match.
Utilize super-resolution fluorescence microtechnic, can, when obtaining nanometer resolution, the evolution of biomacromolecule and organelle micro-structure be monitored continuously, and do not affect its biologically active.Super-resolution fluorescence microtechnic will promote the fast development in the fields such as biomedicine.
At present, the microscopical resolution of super-resolution fluorescence detects main or uses fluorescent nanosphere, but the process of the method is comparatively loaded down with trivial details.Quantum dot is a kind of novel fluorescence nano particle, compare with common fluorescein there is high-fluorescence quantum yield, stable luminescence, exciting light spectrum width, the good fluorescent characteristic such as emission spectrum is narrow, fluorescence is tunable, be widely used in the aspects such as sensor and biomarker.
Summary of the invention
The manufacture method that the object of this invention is to provide a kind of super-resolution fluorescence microscopic system resolution test on-gauge plate, the defect existing to overcome fluorescent nanosphere, and obtain good fluorescent characteristic.
In order to achieve the above object, the technical solution adopted in the present invention is:
The manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: comprise the following steps:
(1), at substrate surface, form photoresist rete;
(2), by design transfer to photoresist rete;
(3), utilize charged polyelectrolytes polymkeric substance to carry out selective adsorption to substrate, in photoresist rete, form after multi-layer quantum point Nano-structure structure, remove photoresist rete;
(4), at quantum dot nano linear surface spin coating polymer layer or other protective seams.
The manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: comprise the following steps:
(1), at substrate surface, form successively the first metallic diaphragm, the second metallic diaphragm and photoresist rete;
(2), by design transfer to photoresist rete;
(3), modify the second metallic diaphragm, in photoresist rete, form after multi-layer quantum point Nano-structure structure, remove photoresist rete;
(4), at quantum dot nano linear surface spin coating polymer layer or other protective seams.
The manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: comprise the following steps:
(1), at substrate surface, form successively the first metallic diaphragm, the second metallic diaphragm, SiO
2rete and photoresist rete;
(2), by design transfer to after on photoresist rete, make mask layer moving on figuratum photoresist rete, remove photoresist;
(3), by design transfer to SiO
2after rete, remove mask layer;
(4), modify the second metal level, at SiO
2in rete, form multi-layer quantum point Nano-structure structure;
(5), at SiO
2multi-layer quantum point Nano-structure body structure surface spin coating polymer layer or other protective seams on rete.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: adopt Polyelectrolyte as polyelectrolyte polymers.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: described SiO
2thicknesses of layers, between 50nm-150nm, utilizes plasma reinforced chemical vapour deposition technology to be formed on the second metallic diaphragm; Described mask layer is Cr film, and thickness is 20nm, utilizes electron beam evaporation technique and stripping technology to be produced on and moves on figuratum photoresist rete.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: can utilize plasma etching technology etching SiO
2rete, and utilize Cr corrosive liquid to remove Cr mask layer.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: described the first metallic diaphragm is 1nm Cr film, and the second metallic diaphragm is 5nm Au film, is all to utilize electron beam evaporation technique to be formed in substrate.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: adopt mercaptan as dressing agent, modify the second metallic diaphragm, make its charge type contrary with quantum dot surface charge.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, it is characterized in that: described base material is Si or quartz, the thickness of described photoresist rete, between 50nm-150nm, is controlled the steepness of photoresist film layer linear by optimizing accelerating potential, exposure dose and development conditions.
The manufacture method of described super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: utilize electron beam lithography by design transfer to photoresist rete, the equidistant linear that the pattern on photoresist rete is 20nm-100nm; Utilize Layer-by-layer package technique, form multi-layer quantum point Nano-structure structure, quantum dot nano linear is between 3-10 layer; At quantum dot nano linear surface spin coating polymer layer or other protective seams, its thickness is hundred nano-scale, does not affect fluorescence excitation and detection.
Compared with prior art, the present invention utilizes nanofabrication technique and self-assembly fabrication techniques fluorescence quantum lines, as fluorescence super-resolution on-gauge plate, can be directly used in the test of super-resolution system, and method for making is simple and reliable.Compare with common fluorescein, the good fluorescent characteristic such as quantum dot has high-fluorescence quantum yield, stable luminescence, exciting light spectrum width, emission spectrum is narrow, fluorescence is tunable, has promoted the precision detecting.
Accompanying drawing explanation
Fig. 1 a to Fig. 1 c is the concrete structure schematic diagram that the present invention makes the first method of fluorescence super-resolution on-gauge plate.
Fig. 2 a to Fig. 2 c is the concrete structure schematic diagram that the present invention makes the second method of fluorescence super-resolution on-gauge plate.
Fig. 3 a to Fig. 3 e is the concrete structure schematic diagram that the present invention makes the third method of fluorescence super-resolution on-gauge plate.
Embodiment
Innovation point of the present invention is: utilize the fluorescence quantum lines of nanofabrication technique and self-assembly fabrication techniques 20nm-100nm live width as fluorescence super-resolution on-gauge plate, can be directly used in super-resolution system testing.
The present invention makes the concrete structure schematic diagram of first method of fluorescence super-resolution on-gauge plate as shown in Fig. 1 a to Fig. 1 c, comprises the following steps:
At the surperficial spin coating photoresist layer 104 of substrate 100, substrate 100 can be Si or quartz;
As shown in Figure 1a, utilize electron beam lithography on photoresist layer 104, to form the equidistant photoresist lines structure of 20nm-100nm, substrate 100 is exposed, the thickness of electron beam resist, between 50nm-150nm, is controlled the steepness of photoresist lines structure by Optimizing Technical (accelerating potential, exposure dose, development conditions etc.);
As shown in Figure 1 b, first utilize charged polyelectrolytes polymkeric substance (Polyelectrolyte) in the selective adsorption on substrate 100 surfaces, form 109 layers, make the electrically charged amount of nano based bottom structure surface institute contrary with quantum dot surface charge; Then utilize Layer-by-layer package technique to form multi-layer quantum point Nano-structure structure 107 on 106 layers of surface, quantum Nano-structure structure 107 is between 3-10 layer;
The Nano-structure structure photoresist of removing photoresist layer 104, forms independently quantum dot nano linear;
As shown in Fig. 1 c, at 107 layers of surperficial spin on polymers layer or other protective seams 108, the thickness of polymeric layer or other protective seams 108 is hundred nano-scale, does not affect fluorescence excitation and detection, to improve the long-time stability of fluorescence super-resolution on-gauge plate.
The present invention makes the schematic flow sheet of second method of fluorescence super-resolution on-gauge plate as shown in Fig. 2 a to Fig. 2 c, comprises the following steps:
As shown in Figure 2 a, utilize electron beam evaporation technique at substrate 100 surfaces successively evaporation the first metallic diaphragm 101 and the second metallic diaphragm 102, substrate 100 can be Si or quartz, the first metallic diaphragm 101 is Cr film, thickness is 1nm, and the second metallic diaphragm 102 is Au layer, and thickness is 5nm;
Then at the surperficial spin coating photoresist layer 104 of the second metallic diaphragm 102, utilize electron beam lithography on photoresist layer 104, to form the equidistant photoresist lines structure of 20nm-100nm, the second metal level 102 is exposed, the thickness of electron beam resist, between 50nm-150nm, is controlled the steepness of photoresist lines structure by Optimizing Technical (accelerating potential, exposure dose, development conditions etc.);
As shown in Figure 2 b, utilize dressing agent mercaptan (Thiol) to modify the second metal level 102 surfaces and form 106 layers, make its charge type contrary with quantum dot surface charge; Utilize Layer-by-layer package technique to form multi-layer quantum point Nano-structure structure 107 on 106 layers of surface, quantum Nano-structure structure 107 is between 3-10 layer;
The Nano-structure structure photoresist of removing photoresist layer 104, forms independently quantum dot nano linear;
As shown in Figure 2 c, at 107 layers of surperficial spin on polymers layer or other protective seams 108, the thickness of polymeric layer or other protective seams 108 is hundred nano-scale, does not affect fluorescence excitation and detection, to improve the long-time stability of fluorescence super-resolution on-gauge plate.
The present invention makes the schematic flow sheet of the third method of fluorescence super-resolution on-gauge plate as shown in Fig. 3 a to Fig. 3 e, comprises the following steps:
As shown in Figure 3 a, utilize electron beam evaporation technique at substrate 100 surfaces successively evaporation the first metallic diaphragm 101 and the second metallic diaphragm 102, substrate 100 can be Si or quartz, the first metallic diaphragm 101 is Cr film, thickness is 1nm, and the second metallic diaphragm 102 is Au layer, and thickness is 5nm;
Then utilize PECVD technology at the SiO of the second metallic diaphragm 102 superficial growth different-thickness
2layer 103, at SiO
2layer 103 surperficial spin coating photoresist layer 104, SiO
2the thickness of layer 103 is between 50nm-150nm, for controlling the thickness of final quantum dotted line bar;
Utilize electron beam lithography on photoresist layer 104, to form the equidistant photoresist lines structure of 20nm-100nm;
As shown in Figure 3 b, utilize Lift-off technique at SiO
2layer 103 surface form mask layer 105, and mask layer 105 is Cr film;
As shown in Figure 3 c, take Cr film as mask, utilize ICP lithographic technique at SiO
2layer 103 forms SiO
2nanometer channel structure, is etched in and etches into the second metal level 102 and stop, and then erodes remaining Cr film, and the second metal level 102 is exposed, and adjusts the technological parameter of ICP lithographic technique, to control SiO
2the steepness of nanometer channel structure;
As shown in Figure 3 d, utilize dressing agent mercaptan (Thiol) to modify the second metal level 102 surfaces and form 106 layers, make its charge type contrary with quantum dot surface charge; Utilize Layer-by-layer package technique to form multi-layer quantum point Nano-structure structure 107 on 106 layers of surface, quantum Nano-structure structure 107 is between 3-10 layer;
As shown in Figure 3 e, at 107 layers of surperficial spin on polymers layer or other protective seams 108, the thickness of polymeric layer or other protective seams 108 is hundred nano-scale, does not affect fluorescence excitation and detection, to improve the long-time stability of fluorescence super-resolution standard edition.
In sum, the present invention utilizes nanofabrication technique and self-assembly fabrication techniques fluorescence quantum lines as fluorescence super-resolution on-gauge plate, can be directly used in super-resolution system testing, and method for making is simple and reliable, and fluorescent characteristic is good.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of making, be equal to replacement, improvement etc., within all should being included in the scope of protection of the invention.
Claims (10)
1. the manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: comprise the following steps:
1) at substrate surface, form photoresist rete;
2) by design transfer to photoresist rete;
3) utilize charged polyelectrolytes polymkeric substance to carry out selective adsorption to substrate, in photoresist rete, form after multi-layer quantum point Nano-structure structure, remove photoresist rete;
4) at quantum dot nano linear surface spin coating polymer layer or other protective seams.
2. the manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: comprise the following steps:
1), at substrate surface, form successively the first metallic diaphragm, the second metallic diaphragm and photoresist rete;
2) by design transfer to photoresist rete;
3) modify the second metallic diaphragm, in photoresist rete, form after multi-layer quantum point Nano-structure structure, remove photoresist rete;
4) at quantum dot nano linear surface spin coating polymer layer or other protective seams.
3. the manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate, is characterized in that: comprise the following steps:
1) at substrate surface, form successively the first metallic diaphragm, the second metallic diaphragm, SiO
2rete and photoresist rete;
2) by design transfer to after on photoresist rete, make mask layer moving on figuratum photoresist rete, remove photoresist;
3) by design transfer to SiO
2after rete, remove mask layer;
4) modify the second metal level, at SiO
2in rete, form multi-layer quantum point Nano-structure structure;
5) at SiO
2multi-layer quantum point Nano-structure body structure surface spin coating polymer layer or other protective seams on rete.
4. the manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate according to claim 1, is characterized in that: adopt Polyelectrolyte as polyelectrolyte polymers.
5. the manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate according to claim 3, is characterized in that: described SiO
2thicknesses of layers, between 50nm-150nm, utilizes plasma reinforced chemical vapour deposition technology to be formed on the second metallic diaphragm; Described mask layer is Cr film, and thickness is 20nm, utilizes electron beam evaporation technique and stripping technology to be produced on and moves on figuratum photoresist rete.
6. the manufacture method of super-resolution fluorescence microscopic system resolution test on-gauge plate according to claim 5, is characterized in that: can utilize plasma etching technology etching SiO
2rete, and utilize Cr corrosive liquid to remove Cr mask layer.
7. according to the manufacture method of the super-resolution fluorescence microscopic system resolution test on-gauge plate described in claim 2 or 3, it is characterized in that: described the first metallic diaphragm is 1nmCr film, the second metallic diaphragm is 5nmAu film, is all to utilize electron beam evaporation technique to be formed in substrate.
8. according to the manufacture method of the super-resolution fluorescence microscopic system resolution test on-gauge plate described in claim 2 or 3, it is characterized in that: adopt mercaptan as dressing agent, modify the second metallic diaphragm, make its charge type contrary with quantum dot surface charge.
9. according to the manufacture method of the super-resolution fluorescence microscopic system resolution test on-gauge plate described in claim 1 or 2 or 3, it is characterized in that: described base material is Si or quartz, the thickness of described photoresist rete, between 50nm-150nm, is controlled the steepness of photoresist film layer linear by optimizing accelerating potential, exposure dose and development conditions.
10. according to the manufacture method of the super-resolution fluorescence microscopic system resolution test on-gauge plate described in claim 1 or 2 or 3, it is characterized in that: utilize electron beam lithography by design transfer to photoresist rete, the equidistant linear that the pattern on photoresist rete is 20nm-100nm; Utilize Layer-by-layer package technique, form multi-layer quantum point Nano-structure structure, quantum dot nano linear is between 3-10 layer; At quantum dot nano linear surface spin coating polymer layer or other protective seams, its thickness is hundred nano-scale, does not affect fluorescence excitation and detection.
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Cited By (9)
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| CN103954600A (en) * | 2014-05-12 | 2014-07-30 | 国家纳米科学中心 | Fluorescent nano-scale component and manufacturing method thereof |
| CN104406944A (en) * | 2014-12-18 | 2015-03-11 | 重庆大学 | Method for increasing optical microscopy imaging resolution by utilizing silicon nanoparticles |
| CN105203508A (en) * | 2015-09-01 | 2015-12-30 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of fluorescent nanometer standard board |
| CN105241635A (en) * | 2015-09-01 | 2016-01-13 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of fluorescent nanometer standard plate used for testing resolution |
| CN107941346A (en) * | 2017-11-16 | 2018-04-20 | 中国电子科技集团公司第十三研究所 | Spatial resolution calibrating installation and preparation method |
| CN108519362A (en) * | 2018-05-08 | 2018-09-11 | 铜仁学院 | A kind of PARA FORMALDEHYDE PRILLS(91,95) realizes the novel nano composite construction chip quickly detected and preparation method |
| CN110095441A (en) * | 2019-04-19 | 2019-08-06 | 中国科学院苏州生物医学工程技术研究所 | A kind of fluorescence nano scale member and its preparation and application |
| CN110108678A (en) * | 2019-04-19 | 2019-08-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of fluorescence nano on-gauge plate and its preparation and application |
| CN112129788A (en) * | 2020-09-16 | 2020-12-25 | 南京大学 | Super-resolution microscale based on quantum dot and DNA origami nano-assembly structure |
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| CN103954600A (en) * | 2014-05-12 | 2014-07-30 | 国家纳米科学中心 | Fluorescent nano-scale component and manufacturing method thereof |
| CN104406944A (en) * | 2014-12-18 | 2015-03-11 | 重庆大学 | Method for increasing optical microscopy imaging resolution by utilizing silicon nanoparticles |
| CN104406944B (en) * | 2014-12-18 | 2017-09-29 | 重庆大学 | The method that optical microphotograph imaging resolution is improved using silicon nano |
| CN105203508A (en) * | 2015-09-01 | 2015-12-30 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of fluorescent nanometer standard board |
| CN105241635A (en) * | 2015-09-01 | 2016-01-13 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of fluorescent nanometer standard plate used for testing resolution |
| CN105203508B (en) * | 2015-09-01 | 2018-09-14 | 中国科学院苏州生物医学工程技术研究所 | The preparation method of fluorescence nano on-gauge plate |
| CN107941346A (en) * | 2017-11-16 | 2018-04-20 | 中国电子科技集团公司第十三研究所 | Spatial resolution calibrating installation and preparation method |
| CN108519362A (en) * | 2018-05-08 | 2018-09-11 | 铜仁学院 | A kind of PARA FORMALDEHYDE PRILLS(91,95) realizes the novel nano composite construction chip quickly detected and preparation method |
| CN110095441A (en) * | 2019-04-19 | 2019-08-06 | 中国科学院苏州生物医学工程技术研究所 | A kind of fluorescence nano scale member and its preparation and application |
| CN110108678A (en) * | 2019-04-19 | 2019-08-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of fluorescence nano on-gauge plate and its preparation and application |
| CN110095441B (en) * | 2019-04-19 | 2021-12-10 | 中国科学院苏州生物医学工程技术研究所 | Fluorescent nanometer scale component and preparation and application thereof |
| CN112129788A (en) * | 2020-09-16 | 2020-12-25 | 南京大学 | Super-resolution microscale based on quantum dot and DNA origami nano-assembly structure |
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