A multilayer structure for fluorescence microscopy imaging, a method of preparation and use thereof

Abstract

The application provides a multilayer structure for fluorescence microscopic imaging, a preparation method and application thereof, wherein the preparation method comprises the following steps: S1, dispersing fluorescent probe molecules into a dispersion solvent, then attaching to a substrate surface and solidifying to obtain a fluorescent probe molecule layer; S2, attaching a coupling agent to the surface of the fluorescent probe molecule layer and solidifying to obtain a coupling layer; S3, patterning the coupling agent on the top to obtain an imaging target, and the imaging target is a multilayer structure. The characteristics of the uniform monolayer film formed by the self-assembly of the fluorescent molecules at the liquid / gas interface are utilized, so that the fluorescent layer and the coupling agent are each a layer, the clustering problem of the fluorescent particles in the coupling agent is avoided, then the photolithography technology is combined for patterning design, and the fluorescence imaging under the fluorescence microscope is realized.

Description

Technical Field

[0001] This invention relates to the field of fluorescence microscopy imaging technology, specifically to a multilayer structure for fluorescence microscopy imaging, its preparation method, and its application. Background Technology

[0002] In the semiconductor field, measurement tools such as electron microscopes (EM), atomic force microscopes (AFM), and scanning tunneling microscopes (STM) are widely used to obtain high-resolution patterns. However, these methods require high vacuum conditions and have drawbacks such as small field of view and low throughput. In contrast, fluorescence microscopy offers advantages such as fast imaging speed, wide field of view, and non-destructive imaging. In the past few years, its resolution has been significantly improved, reaching the tens of nanometers level.

[0003] Current fluorescent small molecules exhibit several limitations, including poor photostability, insufficient fluorescence flicker, and low biocompatibility. Among these, novel fluorescent nanoparticles, upconversion nanoparticles (UCNPs), possess high brightness and photostability, effectively overcoming many limitations of traditional fluorescent probes. UCNPs typically range in size from 10 to 100 nanometers and can convert two or more photons into a single, higher-energy photon. This unique luminescent property allows UCNPs to overcome problems such as photobleaching, flickering, background autofluorescence, and phototoxicity in optical imaging processes, enabling long-term observation in solid-state environments. However, research on the patterning processes for these materials is currently very limited.

[0004] In existing technologies, researchers attach fluorescent probe molecules and coupling agents as a blend to the substrate surface. In this case, the fluorescent probe molecules tend to aggregate and overlap, forming bright spots and causing uneven background illumination, which affects the imaging quality of fluorescence microscopy. Because the particles aggregate and overlap in the z-direction, the fluorescence microscope cannot capture fluorescent probe molecules with single-molecule scintillation properties, thus reducing the accuracy of pattern localization, especially at the template edges. As the critical image size decreases, the impact of these non-ideal phenomena on actual image linewidth measurement becomes more pronounced. This leads to significant linewidth measurement errors, significantly limiting the imaging resolution and preventing it from exceeding the 200nm diffraction limit, thus failing to achieve true super-resolution.

[0005] Figure 3 This is a bright-field one-dimensional line graph of a 200nm hybrid system with a duty cycle of 3:1, based on existing technology. (Example:) Figure 3 As shown, bright-field one-dimensional lines with a duty cycle of 3:1 at 200nm are difficult to distinguish. Summary of the Invention

[0006] This invention is made to solve the above-mentioned problems, and aims to provide a multilayer structure for fluorescence microscopy imaging, its preparation method and its application.

[0007] This invention provides a method for preparing a multilayer structure for fluorescence microscopy imaging, characterized by the following steps: S1, dispersing fluorescent probe molecules in a dispersion solvent, then attaching them to a substrate surface and curing them to obtain a fluorescent probe molecule layer; S2, attaching a coupling agent to the surface of the fluorescent probe molecule layer and curing it to obtain a coupling layer; S3, patterning the coupling agent on top to obtain an imaging target, wherein the imaging target is a multilayer structure.

[0008] The method for preparing a multilayer structure for fluorescence microscopy provided by the present invention may also have the following features: wherein the concentration of the fluorescent probe molecules in the dispersion solvent is 0.1 mmol-10 mmol, the fluorescent probe molecule layer is a single layer or multiple layers, and the fluorescent probe molecule layer and the coupling layer are each a single layer and do not mix with each other.

[0009] The method for preparing a multilayer structure for fluorescence microscopy provided by the present invention may also have the following features: wherein, in step S1, the method of attaching the fluorescent probe molecule layer to the substrate surface includes: the fluorescent probe molecules being placed on the substrate surface by drop casting and then cured; or, the fluorescent probe molecules forming a self-assembled layer at the gas and liquid interface and transferring it to the substrate surface and then curing it; or, the fluorescent probe molecules specifically enriching on the substrate surface in a liquid phase environment and then curing it; or, the fluorescent probe molecules dispersing probe molecules into a monolayer at the gas-liquid interface and transferring it to the substrate.

[0010] The method for preparing a multilayer structure for fluorescence microscopy provided by the present invention may also have the following feature: when the fluorescent probe molecules are placed on the substrate surface by drop casting, the fluorescent probe molecules are well wetted with the substrate.

[0011] The method for preparing a multilayer structure for fluorescence microscopy provided by the present invention may also have the following feature: wherein the patterning preparation step is photolithography development, and the coupling agent is photoresist.

[0012] The method for preparing a multilayer structure for fluorescence microscopy provided by the present invention may also have the following feature: wherein the photoresist does not chemically react with the fluorescent probe molecules.

[0013] The method for preparing multilayer structures for fluorescence microscopy provided by the present invention may also have the following feature: a solvent and a developing solution are used in conjunction with the photolithography development, and the solvent, the developing solution and the fluorescent probe molecules do not undergo chemical reactions.

[0014] The method for preparing multilayer structures for fluorescence microscopy provided by the present invention may also have the following feature: wherein the fluorescent probe molecules maintain random scintillation in a solid environment.

[0015] The present invention also provides a multilayer structure for fluorescence microscopy imaging, which is characterized by being obtained by the above-described method for preparing a multilayer structure for fluorescence microscopy imaging.

[0016] The present invention also provides an application of a multilayer structure for fluorescence microscopy imaging, characterized by performing fluorescence microscopy imaging on the imaging target.

[0017] The role and effect of invention

[0018] According to the multilayer structure, preparation method, and application of fluorescence microscopy imaging disclosed in this invention, the property of fluorescent molecules self-assembling at the liquid / gas interface to form a uniform monolayer film is utilized, ensuring that the fluorescent layer and the coupling agent are each a separate layer, thus avoiding the clustering problem of fluorescent particles in the coupling agent. Subsequently, patterning design is performed using photolithography technology to achieve fluorescence imaging under a fluorescence microscope. The method of this invention utilizes particles with fluorescent scintillation properties to generate a fluorescence intensity contrast between exposed and unexposed areas, thereby achieving precise pattern measurement. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the multi-layer structure in an embodiment of the present invention;

[0020] Figure 2 This is a bright-field one-dimensional line graph of the bilayer film system in Embodiment 1 of the present invention with a duty cycle of 3:1 at 200 nm; and

[0021] Figure 3 It is a bright-field one-dimensional line graph of the existing hybrid system with a duty cycle of 3:1 at 200nm. Detailed Implementation

[0022] To make the technical means, creative features, objectives and effects of this invention easy to understand, the following embodiments, in conjunction with the accompanying drawings, specifically illustrate the multilayer structure, preparation method and application of this invention for fluorescence microscopy.

[0023] Figure 1 This is a schematic diagram of the multi-layer structure in an embodiment of the present invention.

[0024] like Figure 1 As shown, the preparation method of the multilayer structure 100 for fluorescence microscopy imaging in this embodiment includes the following steps:

[0025] S1, fluorescent probe molecules are attached to the surface of substrate 10 and cured to obtain fluorescent probe molecule layer 20.

[0026] The concentration of the fluorescent probe molecules in the dispersion solvent is 0.1 mmol to 10 mmol. The fluorescent probe molecules form a uniform distribution in the dispersion solvent, and the cluster size is not larger than the required minimum size.

[0027] Methods for attaching fluorescent probe molecular layer 20 to the surface of substrate 10 include:

[0028] Fluorescent probe molecules are deposited on the substrate surface using a drop casting method and then cured. The fluorescent probe molecules are well wetted with the substrate.

[0029] Fluorescent probe molecules form a self-assembled layer at the gas-liquid interface, transfer to the substrate surface, and solidify.

[0030] Fluorescent probe molecules are specifically enriched on the substrate surface and solidified in a liquid environment.

[0031] Fluorescent probe molecules are dispersed into a monolayer at the gas-liquid interface and transferred to the substrate 10.

[0032] S2, the coupling agent is attached to the surface of the fluorescent probe molecular layer and cured to obtain the coupling layer 30.

[0033] The fluorescent probe molecular layer 20 can be a single layer or multiple layers. The fluorescent probe molecular layer 20 and the coupling layer 30 are each a single layer and do not mix with each other.

[0034] S3, pattern the top coupling layer 30 to obtain the imaging target, which is a multilayer structure 100.

[0035] The patterning process involves photolithography and development, and the coupling agent is photoresist. The photoresist does not chemically react with the fluorescent probe molecules.

[0036] The development process in photolithography uses a solvent and a developer solution, and the solvent, the developer solution, and the fluorescent probe molecules do not undergo chemical reactions.

[0037] Fluorescent probe molecules maintain random flashing in a solid environment.

[0038] In this embodiment, a monolayer of UCNPs is formed using the self-assembly properties of UCNPs at the DEG / air interface, and then transferred onto a silicon substrate. PMMA is subsequently coated, and patterning is achieved using EBL. UCNPs dissolved in cyclohexane are dropped onto the DEG surface, and the cyclohexane is allowed to evaporate over several minutes. To transfer the monolayer film floating on the liquid surface onto the silicon wafer, the silicon wafer beneath the film is lifted very slowly. A low-temperature drying technique is used to control the evaporation rate of the DEG, ultimately forming a uniform UCNP self-assembled monolayer (SAM) film across the entire silicon wafer.

[0039] The present invention also provides an application of the multilayer structure 100 described above for fluorescence microscopy imaging, for performing fluorescence microscopy imaging on the imaging target.

[0040] Figure 2 This is a bright-field one-dimensional line graph of the bilayer film system with a duty cycle of 3:1 at 200nm in Embodiment 1 of the present invention.

[0041] like Figure 2 As shown, this invention utilizes STORM to analyze wide-field images. STORM is a specific type of single-molecule localization microscopy, allowing structural resolution beyond the diffraction limit. The positions of individual molecules were determined after image filtering using wavelet filters (3rd order, 2nd order). The STORM algorithm employs three key algorithmic functions: centroid determination of connected components, non-maximum suppression, and local maxima detection. This invention establishes a threshold of 0.4 times the standard deviation (0.4*std, Wave.F1) and employs local maxima testing to improve detection accuracy. Since the experimental localization locations exhibit a Gaussian distribution, the half-maximum full width (FWHM) and peak spacing are used to evaluate the reconfigured dimensions.

[0042] Example 2

[0043] In this embodiment, compared with Embodiment 1, other small molecule fluorescent probes are co-mixed into the coupling agent in step S2, followed by a co-localization process involving two super-resolution reconstructions.

[0044] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a multilayer structure for fluorescence microscopy imaging, characterized in that, Includes the following steps: S1, the fluorescent probe molecules are dispersed in a dispersion solvent, then attached to the substrate surface and cured to obtain a fluorescent probe molecule layer; S2, the coupling agent is attached to the surface of the fluorescent probe molecular layer and cured to obtain the coupling layer; S3, The top coupling layer is patterned to obtain the imaging target, which has a multi-layer structure. The fluorescent probe molecular layer and the coupling layer are each a single layer and do not mix. The patterning process is photolithography, the coupling agent is photoresist, the photoresist does not chemically react with the fluorescent probe molecules, and the photolithography development process uses a solvent and a developer solution, the solvent, the developer solution and the fluorescent probe molecules do not chemically react.

2. The method for preparing a multilayer structure for fluorescence microscopy imaging according to claim 1, characterized in that: in, The fluorescent probe molecular layer can be a single layer or multiple layers.

3. The method for preparing a multilayer structure for fluorescence microscopy according to claim 1, Its features are: In step S1, the method of attaching the fluorescent probe molecular layer to the substrate surface includes: The fluorescent probe molecules are placed on the substrate surface by drop casting and then solidified; or, The fluorescent probe molecules form a self-assembled layer at the gas-liquid interface, transfer to the substrate surface, and solidify; or, The fluorescent probe molecules specifically accumulate on the substrate surface in a liquid phase environment and then solidify; or, The fluorescent probe molecules are dispersed into a monolayer at the gas-liquid interface and then transferred to the substrate.

4. The method for preparing a multilayer structure for fluorescence microscopy imaging according to claim 3, characterized in that: in, When the fluorescent probe molecules are placed on the substrate surface by drop casting, the fluorescent probe molecules are well wetted with the substrate.

5. The method for preparing a multilayer structure for fluorescence microscopy imaging according to claim 1, characterized in that: in, The fluorescent probe molecules maintain random flashing in a solid state.

6. A multilayer structure for fluorescence microscopy imaging, characterized in that, The multilayer structure for fluorescence microscopy is obtained by any one of the preparation methods of claims 1 to 5.

7. An application of the fluorescence microscopy imaging multilayer structure as described in claim 6, characterized in that: Fluorescence microscopy was performed on the imaging target.

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