CN114265131A

CN114265131A - Biological fluid microsphere, preparation method and optical imaging method of microsphere lens

Info

Publication number: CN114265131A
Application number: CN202111543898.3A
Authority: CN
Inventors: 杨慧; 陈希; 顾国强
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-04-01
Anticipated expiration: 2041-12-16
Also published as: WO2023109703A1; CN114265131B

Abstract

According to the biological liquid microsphere and the preparation method thereof, biological liquid is coated on the convex column of PDMS, egg white on the convex column is transferred to the liquid layer of the PDMS, the egg white is self-assembled in the PDMS to form microspheres, and the PDMS is removed to obtain the biological liquid microsphere. In addition, the application also provides an optical imaging method based on the biological fluid microsphere.

Description

Biological fluid microsphere, preparation method and optical imaging method of microsphere lens

Technical Field

The invention relates to the technical field of medical instruments, in particular to a biological liquid microsphere, a preparation method of the biological liquid microsphere and an optical imaging method of a microsphere lens.

Background

The optical microscopic imaging technology plays an important role in the process of observing the real world and discovering and exploring the unknown world. Limited by the diffraction limit of light, the resolution of conventional optical microscopes is no less than 200nm at most when the wavelength of the light source used is in the visible range. The reason for the diffraction limit is that evanescent waves propagate only very close to the near field, with intensity decaying exponentially with increasing distance, and with high spatial frequency signals representing fine structural information of the object.

The super-resolution imaging technology can cross or bypass the diffraction limit barrier, and the imaging of the object with the sub-diffraction limit scale is realized. Imaging techniques include Near-field optical microscopy (NSOM), and far-field super-resolution imaging techniques (e.g., stimulated radiation depletion microscopy, STED), photosensitive positioning microscopy (PALM), etc.). The observation and detection of viruses, proteins, intracellular fine structures and the like can be realized by using the technologies, and the observation and detection are important and hot spots in the research of modern optical imaging technologies.

However, these techniques either require expensive and complicated equipment or require fluorescent labeling of the sample, which causes unavoidable phototoxicity, and thus have been greatly limited in label-free and live cell imaging. In the last decade, a novel microsphere lens-assisted super-resolution imaging technology discovered by scientific researchers of multiple subject groups at home and abroad combines microspheres with a conventional optical microscope, and utilizes the super-focusing optical field effect of the microspheres to obtain the resolution and imaging of sub-diffraction limit scale objects. The super-resolution imaging technology has the characteristics and advantages of no mark, no phototoxicity, low manufacturing cost, high imaging speed, simple device structure and the like.

The microspheres used in current super-resolution imaging are mostly inorganic and organic compounds, such as SiO₂Microspheres, BaTiO₃Glass microspheres, polystyrene microspheres, etc., have poor compatibility with biological samples, limiting the application of this technique in biological and medical imaging. The egg white has good biocompatibility and bioactivity, is easy to absorb and degrade by human bodies, has wide sources and low price, is simple and feasible in preparation process, and has no similar report in the prior art when the microspheres taking the egg white as a raw material are applied to super-resolution optical imaging.

Disclosure of Invention

In view of the above, there is a need to provide a bio-fluid microsphere for performing super-resolution imaging observation with a bio-fluid microsphere array structure.

In order to solve the problems, the invention adopts the following technical scheme:

one of the purposes of the application is to provide a preparation method of biological fluid microspheres, which comprises the following steps:

coating biological fluid on the convex column of PDMS, wherein the biological fluid comprises egg white;

transferring egg white on the posts onto the liquid layer of PDMS;

the egg white is self-assembled in the PDMS to form microspheres;

and removing the PDMS to obtain the biological fluid microspheres.

In some embodiments, the step of coating the egg white solution on the PDMS convex pillars specifically includes the following steps:

and transferring the egg white solution on the PDMS convex column onto the PDMS liquid layer by means of light pressure.

In some embodiments, in the step of removing the PDMS to obtain the bio-fluid microspheres, the step of removing the PDMS specifically includes: and removing PDMS by using an ethyl acetate solvent to obtain the biological fluid microspheres.

The second purpose of the application is to provide a biological fluid microsphere, which is prepared by the preparation method of the biological fluid microsphere, and the diameter of the biological fluid microsphere is between 5 and 500 um.

The third objective of the present application provides an optical imaging method of a microsphere lens, comprising the following steps:

directly stripping the protective film on the surface of the blue-ray disc;

coating a PDMS film on the surface of the Blu-ray disc;

transferring the egg white on the convex columns to the PDMS film;

and imaging the grating structure of the blue-ray disc by using the biological liquid microspheres.

In some embodiments, the step of peeling off the protective film on the surface of the blu-ray disc specifically includes the following steps:

the blue-ray disc is placed on a heating plate with the temperature of 45-55 ℃ for heating treatment for 1.5-2.5 min, and then the protective film on the surface is torn off by using tweezers.

In some embodiments, in an optical imaging method of a microsphere lens, the method specifically includes: the thickness of the PDMS film is 200-; the egg white liquid drops can self-assemble to form an array distribution microsphere structure through surface tension and interface interaction with PDMS.

In some of these embodiments, the grating structure is a periodic stripe structure consisting of lines with a line width of 180nm and an arrangement of grooves with a pitch of 140 nm.

By adopting the technical scheme, the invention has the following technical effects:

according to the biological liquid microsphere and the preparation method thereof, the biological liquid is coated on a convex column of PDMS, egg white on the convex column is transferred to a liquid layer of the PDMS, the egg white is self-assembled in the PDMS to form the microsphere, and the PDMS is removed to obtain the biological liquid microsphere.

In a second aspect, the application further provides an optical imaging method of the microsphere lens, the protective film on the surface of the optical disc is peeled off, the PDMS film is coated on the surface of the blu-ray disc, the egg white on the convex columns is transferred to the PDMS film, and the grating structure of the blu-ray disc is imaged by using the biological liquid microspheres. The thickness of the PDMS film is 200-; the egg white liquid drops are self-assembled through surface tension and the interface interaction between the egg white liquid drops and PDMS to form a microsphere structure distributed in an array; the grating structure is a periodic stripe structure formed by arranging lines with the line width of 180nm and grooves with the space of 140nm, the stripes of the blue-ray disc amplified by the single-layer egg white microstructure are observed through a microscope, the imaging method can realize the resolution of a sample with the sub-diffraction limit scale (less than 200nm) under an optical microscope, and the resolution capability exceeding the diffraction limit is displayed.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention or in the description of the prior art will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of the steps for preparing bio-fluid microspheres according to example 1 of the present invention;

FIG. 2 is a schematic view of a microscope of biological fluid microspheres provided in example 1 of the present invention;

FIG. 3 is a flowchart illustrating the steps of a method for optically imaging a microsphere lens provided in example 2 of the present application;

fig. 4 is a schematic view of super-resolution imaging of the microsphere lens provided in example 2 of the present application.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In the description of the present invention, it is to be understood that the terms "upper", "lower", "horizontal", "inside", "outside", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.

Example 1

Referring to fig. 1, a flowchart of steps for preparing bio-fluid microspheres according to an embodiment of the present invention is provided, which includes the following steps:

step S110: coating biological liquid on the PDMS convex column, wherein the biological liquid comprises egg white solution.

In this embodiment, the PDMS convex pillar may be obtained by a photolithography mold, a machining mold (COC, COP, and PMMA mold), a laser engraving mold, and an imprinting method.

Specifically, the egg white solution is coated on the PDMS convex studs, and the contact time is generally 5s according to experience.

Step S120: egg white on the posts was transferred to the liquid layer of PDMS.

In this example, egg white on the posts was transferred to the PDMS liquid layer by light pressure.

Step S130: the egg white self-assembles in the PDMS to form microspheres.

Transferring the egg white liquid drops on the convex columns to the PDMS film, wherein the egg white liquid drops can form a microsphere structure distributed in an array through surface tension and interface interaction between the egg white liquid drops and the PDMS;

it is understood that the egg white droplets will self-assemble into an array of microsphere structures through surface tension and interfacial interaction with PDMS.

Step S140: and removing the PDMS to obtain the biological fluid microspheres.

In this example, PDMS was removed with ethyl acetate solvent to obtain the bio-fluid microspheres.

Please refer to fig. 2, which is a microscope image of the bio-fluid microspheres provided in the present application.

The biological fluid microsphere and the preparation method thereof can be used for preparing a manufacturing technology of microspheres with high circularity from several micrometers to hundreds of micrometers, the whole process is prepared in a stable environment, the preparation method is simple and convenient, and the prepared microspheres have good biocompatibility.

Example 2

Referring to fig. 3, a flowchart of the steps of a method for optically imaging a microsphere lens is provided, which includes the following steps:

step S210: directly stripping the protective film on the surface of the blue-ray disc.

In this embodiment, the Blu-ray disc is placed on a heating plate at 45-55 ℃ for 1.5-2.5 min, and then the surface protective film is removed with tweezers.

Step S220: and coating a PDMS film on the surface of the blue-ray disc, wherein the thickness of the PDMS film is 200-1000 um.

In this embodiment, a PDMS film is spin-coated on the blu-ray disc.

Step S230: transferring the egg white liquid drops on the convex columns to the PDMS film, wherein the egg white liquid drops can form a microsphere structure distributed in an array through surface tension and interface interaction between the egg white liquid drops and the PDMS;

step S240: and imaging the microstructure of the blue-ray disc by using the biological liquid microspheres.

Referring to fig. 4, the egg white array microsphere can be used to observe a grating structure on a blu-ray disc, the grating structure is a periodic stripe structure formed by lines with a line width of 180nm and grooves with a distance of 140nm, and the stripes of the blu-ray disc amplified by a single-layer egg white microstructure are observed through a microscope, so that the imaging method can realize the resolution of a sample with a sub-diffraction limit scale (less than 200nm) under an optical microscope, and shows the resolution capability exceeding the diffraction limit.

The foregoing is considered as illustrative only of the preferred embodiments of the invention, and is presented merely for purposes of illustration and description of the principles of the invention and is not intended to limit the scope of the invention in any way. Any modifications, equivalents and improvements made within the spirit and principles of the invention and other embodiments of the invention without the creative effort of those skilled in the art are included in the protection scope of the invention based on the explanation here.

Claims

1. The preparation method of the biological fluid microspheres is characterized by comprising the following steps of:

coating biological liquid on the PDMS convex column, wherein the biological liquid comprises egg white solution;

transferring the egg white solution on the pillars onto the liquid layer of PDMS;

the egg white solution is self-assembled in the PDMS to form microspheres;

removing the PDMS to obtain the bio-fluid microspheres.

2. The method for preparing biological fluid microspheres according to claim 1, wherein the step of transferring the egg white solution on the posts onto the liquid layer of PDMS comprises the following steps:

3. The method for preparing the bio-fluid microspheres according to claim 1, wherein the step of removing the PDMS to obtain the bio-fluid microspheres specifically comprises:

and removing PDMS by using an ethyl acetate solvent to obtain the biological fluid microspheres.

4. A bio-fluid microsphere, which is prepared by the method for preparing a bio-fluid microsphere according to any one of claims 1 to 2.

5. The biological fluid microsphere of claim 4, wherein the diameter of the biological fluid microsphere is between 5 μm and 500 μm.

6. An optical imaging method of a microsphere lens, comprising the steps of:

stripping the protective film on the surface of the blue-ray disc;

coating a PDMS film on the surface of the Blu-ray disc;

transferring the egg white on the convex columns to the PDMS film;

7. The optical imaging method according to claim 6, wherein the step of peeling the protective film from the surface of the Blu-ray disc comprises the steps of:

8. The optical imaging method according to claim 6, wherein the thickness of the PDMS film is 200-1000 μm.

9. The optical imaging method according to claim 6, wherein the egg white droplets self-assemble into an array of microsphere structures through surface tension and interfacial interaction with PDMS.

10. The optical imaging method according to claim 6, wherein the grating structure is a periodic stripe structure consisting of lines with a line width of 180nm and a groove arrangement with a pitch of 140 nm.