A method of patterned traction force microscopy
By preparing patterned templates on a hard substrate and depositing fluorescent microspheres, combined with passivation and cross-linking treatments, the resolution and equipment cost issues of existing gel patterning technologies are solved, achieving efficient patterned gel imaging suitable for general wide-field microscopy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NANKAI UNIV
- Filing Date
- 2025-04-29
- Publication Date
- 2026-07-14
AI Technical Summary
Existing gel patterning techniques are inadequate in terms of pattern resolution, ease of operation, and equipment cost, making it difficult to achieve efficient imaging using ordinary wide-field microscopes.
Patterned templates were prepared on a hard substrate, and patterned adhesion protein layers were formed by photolithography. After depositing fluorescent microspheres, the patterned structures were transferred to the surface of the gel substrate. Passivating agents and cross-linking agents were used to enhance the binding force of the adhesion proteins and avoid direct mechanical contact. Imaging was performed using a conventional wide-field microscope.
This method achieves a uniform monolayer distribution of fluorescent microbeads on the gel surface, making it suitable for imaging with ordinary wide-field microscopes. It avoids high equipment costs and gel damage caused by mechanical contact, and improves process repeatability and imaging clarity.
Smart Images
Figure CN120594468B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of gel patterning imaging technology, and more particularly to a patterned traction force microscopy imaging method. Background Technology
[0002] Patterned traction microscopy has a wide range of applications in many fields.
[0003] Applications in cell biology: Used to study the mechanical mechanisms of cell migration, cell adhesion, and cell differentiation. For example, researchers have used patterned traction force microscopy to discover the vortex-like dynamic interactions generated by cells during migration, as well as the changing patterns of cell adhesion forces in different microenvironments.
[0004] Applications in tumor research: It helps to reveal the relationship between the mechanical properties of tumor cells and tumor occurrence, development, invasion, and metastasis. For example, by studying the distribution of traction forces on a patterned substrate, it was found that the specific heterogeneous mechanical microenvironment within tumor cell sheets can trigger epithelial-mesenchymal transition, thereby affecting the metastatic ability of tumors.
[0005] Applications in tissue engineering: It can be used to evaluate the mechanical response of cells on biomaterials, providing a basis for optimizing the design of tissue engineering scaffolds to promote cell adhesion, proliferation and tissue formation.
[0006] Gel patterning is a crucial step in patterned traction microscopy, and it mainly includes the following:
[0007] Direct microcontact printing (DMP) involves fabricating polydimethylsiloxane (PDMS) stamps using microfabricated templates. The stamps are then incubated with an activated fibronectin (Fn) solution, leaving a thin Fn layer on the stamp. The stamp is then directly applied to an activated gel. After removing the stamp, the contact area between the gel and the stamp becomes a pattern of cell adhesion. However, this method suffers from drawbacks: mechanical contact during operation can easily damage the gel surface, and the low tolerance for operational errors makes it difficult to achieve high-resolution patterns.
[0008] Indirect microcontact printing technology: This method also utilizes microfabricated templates to create PDMS stamps. After incubating the stamps with an activated fibronectin solution, the stamps are placed on a coverslip, transferring the pattern from the stamps to the coverslip. Finally, a gel is prepared on the coverslip, and after the gel polymerizes, the coverslip is removed to obtain a patterned gel substrate. Although this method avoids direct contact, the pattern resolution is still limited by the precision of the stamp-slip contact.
[0009] Direct photolithography involves preparing a photosensitive gel on a photomask. After the gel polymerizes, deep ultraviolet light with a wavelength of 180 nm is irradiated through the mask. Ozone is generated and activated within the irradiated area of the gel. Fibronectin (Fn) binds only to the activated area, thus forming a pattern. However, this technology requires special photosensitive gel materials, resulting in high equipment costs and a high technical barrier.
[0010] Figure 3 The effects of direct microcontact printing and indirect microcontact printing were demonstrated, but neither achieved satisfactory results in image transfer and imaging.
[0011] Meanwhile, the background noise of the traditional microsphere mixing method is >30%, resulting in insufficient signal-to-noise ratio in wide-field microscopy imaging. It can only be used with confocal microscopy or super-resolution microscopy, which is costly.
[0012] In summary, existing gel patterning technologies have many shortcomings in terms of pattern resolution, ease of operation, and equipment cost. Therefore, the market urgently needs a method that adopts a novel gel patterning scheme and can be efficiently imaged using a conventional wide-field microscope. Summary of the Invention
[0013] The present invention aims to solve at least one of the technical problems existing in the related art.
[0014] Therefore, the present invention provides a patterned traction force microscopic imaging method, the steps of which include:
[0015] a) Fabricating patterned templates on a hard substrate;
[0016] b) Prepare a patterned adhesion protein layer on the patterned template and passivate the non-patterned areas;
[0017] c) Deposit fluorescent microspheres on the surface of the adhesion protein layer to form a uniformly distributed layer of fluorescent microspheres;
[0018] d) Prepare a gel substrate on the patterned template, and transfer the patterned adhesion protein layer and the fluorescent microsphere layer to the surface of the gel substrate to form a patterned gel substrate.
[0019] Further, step a) includes preparing the patterned template on the hard substrate by photolithography, specifically including: spin-coating photoresist, exposing through a mask, and developing to remove the photoresist from the non-patterned areas, wherein the photoresist is a positive photoresist and the spin-coating thickness is 1-2 μm.
[0020] Furthermore, the hard substrate is a circular cover glass, which undergoes plasma cleaning and surface modification treatment before photolithography. The surface modification treatment includes hexamethyldisilazane vapor deposition.
[0021] Further, step b) includes: passivating the unpatterned areas of the patterned template with a passivating agent, depositing adhesive proteins in the patterned areas with adhesive proteins, and applying a crosslinking agent to enhance the binding of the adhesive proteins to the gel substrate.
[0022] Further, the passivating agent is a polylysine-polyethylene glycol copolymer with a concentration of 0.1–1 mg / mL; the adhesion protein is fibronectin with a concentration of 5–50 μg / mL; and the crosslinking agent is a 0.05% glutaraldehyde solution.
[0023] Further, step c) includes: mixing fluorescent microspheres with surfactants and solvents to form a solution, applying it to the surface of the patterned template, and forming the uniformly distributed fluorescent microsphere layer after the solvent evaporates.
[0024] Furthermore, the fluorescent microspheres have a solid content of 1% and a diameter of 0.2 μm; the surfactant is a 0.02% poloxamer solution; and the solvent is ethanol.
[0025] Further, step d) includes: covering the patterned template with a gel prepolymer solution, initiating polymerization to form the gel substrate, separating the gel substrate from the patterned template, and transferring the adhesion protein layer and the fluorescent microsphere layer to the surface of the gel substrate.
[0026] Furthermore, the gel prepolymer liquid includes acrylamide and methylenebisacrylamide, wherein the concentration of acrylamide is 6% to 12% and the concentration of methylenebisacrylamide is 0.03% to 0.3%.
[0027] Furthermore, the gel substrate is attached to a silanized coverslip, the silanization process enhancing the adhesion between the gel substrate and the coverslip.
[0028] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:
[0029] This method enables fluorescent microbeads to be uniformly distributed in a monolayer on the gel surface, making it suitable for imaging with ordinary wide-field microscopes.
[0030] Patterning is an indirect transfer patterning scheme that avoids the stress deformation problem in soft lithography, while also avoiding damage to the gel surface caused by direct mechanical contact, thus improving process repeatability.
[0031] There are no requirements for lithography machines or photoresists, reducing equipment requirements. Deep ultraviolet lithography and special photosensitive gel materials are not needed.
[0032] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description
[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0034] Figure 1 This is a schematic diagram of the photolithography process of the patterned traction force microscopic imaging method of the present invention;
[0035] Figure 2 This is a schematic diagram of the pattern transfer scheme of the patterned traction force microscopic imaging method of the present invention;
[0036] Figure 3 These are effect diagrams of direct microcontact printing and indirect microcontact printing in existing gel patterning technologies;
[0037] Figure 4 These are comparison images of the effects of different processing methods of the patterned traction force microscopic imaging method of the present invention on the fluorescent microspheres on the gel surface;
[0038] Figure 5 This is a patterned effect diagram of the crosslinking of glutaraldehyde in this invention;
[0039] Figure 6 This is a patterned effect diagram of the present invention without the use of glutaraldehyde crosslinking;
[0040] Figure 7 This is a comparison diagram showing the effect of the patterned traction force microscopy imaging method of the present invention on the patterning of gels of different sizes. Detailed Implementation
[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention but should not be used to limit the scope of this invention.
[0042] The following is combined Figures 1 to 7 This paper describes a specific embodiment of a patterned traction force microscopy imaging method based on the present invention.
[0043] The raw materials and components used in this embodiment are as follows:
[0044] Substrate material: 25mm circular cover glass;
[0045] Photoresist: Positive photoresist, spin-coated thickness 1-2 μm;
[0046] Passivating agent: polylysine-polyethylene glycol copolymer (PLL-PEG) solution, concentration 0.1-1 mg / mL;
[0047] Adhesion protein: fibronectin (Fn) solution, concentration 5-50 μg / mL;
[0048] Crosslinking agent: 0.05% glutaraldehyde solution;
[0049] Gel prepolymer solution: Acrylamide (Acr) 6%–12%, methylenebisacrylamide (Bis) 0.03%–0.3%;
[0050] Fluorescent microspheres: 1% solid content, 0.2 μm in diameter;
[0051] Dispersant: 0.02% poloxamer (F127) solution.
[0052] A patterned Fn layer was prepared on a rigid substrate (glass coverslip) and then transferred to the surface of a polyacrylamide gel. The specific implementation process is described below:
[0053] Figure 1 The main process of the photolithography section is shown.
[0054] At room temperature, a 25 mm diameter glass slide was washed out of the acid bottle, dried with an air pump, and then cleaned with a plasma cleaner for 2 min 30 s. Then, it was deposited with hexamethyldisilazane (HMDS) in a sealed vacuum dish for 30 min to form a photolithographic glass slide.
[0055] The photolithography process then proceeds in the photolithography chamber. The specific steps are as follows: 5 drops of photoresist are spin-coated onto the surface of the photolithography glass slide using a spin coater, then heated and cured on a 100°C heating stage for 2 minutes and 30 seconds. The slide is then photolithographically etched for 10 seconds to transfer the pattern from the photomask to the glass slide. After photolithography, the slide is immersed in a developing solution to remove the photoresist from the non-patterned areas. It is then dried using an air pump and finally heated and dried on a 100°C heating stage for 2 minutes and 30 seconds. The slide is then stored in a 100mm dish for long-term preservation.
[0056] Figure 2 The main process of the pattern transfer scheme is shown.
[0057] The Fn pattern on the surface of the photolithographic glass slide serves as a template for subsequent gel patterning. The photolithographic glass slide is used as a cover glass, and polyacrylamide gel is prepared on it. The steps are as follows:
[0058] After removing the glass slide with the photolithographic pattern, treat it with plasma for 5 minutes to remove HMDS from the unpatterned surface.
[0059] Then immerse it in acetone placed on an 80℃ heating table for 5 minutes, and then clean it with ultrasound for 5 minutes to remove the photoresist on the pattern surface. After immersion, use isopropanol to remove excess acetone, and then use deionized water to remove excess isopropanol.
[0060] The unpatterned areas were passivated by rinsing with phosphate-buffered saline (PBS) solution in a steel tank, adding 500 μL PLL-PEG solution to the steel tank, and soaking at room temperature for 30 min. The remaining PLL-PEG was recovered after soaking, and it could be recovered 3 times.
[0061] After rinsing with PBS solution, 500 μL of Fn solution is added to the steel tank and incubated in an incubator for 30 min. After incubation, the remaining Fn is recovered, which can be done 5 times. Then, the tank is rinsed with PBS solution. Finally, an adhesion protein layer is formed on the surface of the photolithography slide.
[0062] Add 500 μL of 0.05% glutaraldehyde solution, soak at room temperature for 5 minutes, rinse with sterile water after soaking, absorb the surface moisture and let it air dry.
[0063] It should be noted that in the process of fabricating patterns on a hard substrate (glass coverslip), the photolithographic slide contains HMDS, which has a strong binding effect with Fn. Direct contact with the gel only results in a small portion of Fn being transferred to the gel, insufficient for cells to properly adhere to the patterned area. Immersing the slide in glutaraldehyde acts as a cross-linking agent, allowing Fn to cross-link with the gel, thereby increasing the amount of Fn transferred and meeting the cell adhesion requirements. Figure 5 As shown, the pattern transfer of Fn is clearer after cross-linking.
[0064] The sedimentation scheme and gel transfer steps for fluorescent microspheres on the gel surface are as follows:
[0065] The surfactant F-127 solution was heated in a bead bath at 37°C until the crystals were completely dissolved. 200 μL of ethanol was added to a 200 μL centrifuge tube, followed by 1 μL of F-127 solution. After mixing well, 1 μL of 1% solids Beads was added to obtain an ethanol solution of Beads. At room temperature, 40 μL of the ethanol solution of Beads was dropped onto the surface of a glass slide. The ethanol evaporated quickly, leaving the Beads adsorbed on the surface of the glass slide.
[0066] It should be noted that conventional traction imaging uses confocal imaging, directly incorporating fluorescent microbeads into the gel. To reduce costs, this design utilizes a standard wide-field microscope for imaging. However, to avoid defocusing noise caused by directly incorporating fluorescent microbeads into the gel, this design covers the gel surface with uniformly distributed fluorescent microbeads. Directly placing dispersed fluorescent microbeads on the slide surface leads to agglomeration after solvent evaporation, severely impacting subsequent imaging.
[0067] After analysis, a combination of ethanol and dispersant was adopted, which can effectively reduce background noise, facilitate the positioning and identification of fluorescent microbeads, and thus improve the accuracy of traction force calculation.
[0068] The patterned layer transfer step includes covering the surface of a photolithographic glass slide with a gel prepolymer, then covering it with a silanized glass slide, terminating the polymerization process, and finally separating the photolithographic glass slide from the gel after the polymerization is complete. Because the gel has strong adhesion to the silanized glass slide, the gel remains on the silanized glass slide, while the patterned Fn layer is transferred from the photolithographic glass slide to the gel surface.
[0069] like Figure 4 The images show the effects of different treatment methods on the surface of fluorescent microspheres. (A) The fluorescent microsphere mixed gel solution exhibits defocusing noise. (B) The aqueous solvent solution with added surfactant shows severe agglomeration of fluorescent microspheres. (C) The solution with added ethanol surfactant shows uniform dispersion of fluorescent microspheres.
[0070] like Figure 5 and Figure 6 As shown, the image compares the effects of using glutaraldehyde for gel patterning with and without glutaraldehyde. The scale bar of the image is 200 μm.
[0071] Without the use of glutaraldehyde crosslinking, the patterned cells struggled to maintain their pattern shape and adhere properly. However, the patterned cells with glutaraldehyde crosslinking exhibited clear and stable patterns, demonstrating that glutaraldehyde enhances the transfer efficiency of Fn during patterning, enabling cells to adhere better.
[0072] In further verification, the patterned traction force microscopy imaging method of the present invention was used to verify various pattern sizes and wide-field microscope imaging effects.
[0073] like Figure 7 As shown, patterns and traction forces of 75μm, 100μm, 150μm, and 200μm are displayed from left to right, with the scale bar in the figure being 20μm.
[0074] Through the verification of the above embodiments, the patterned traction force microscopy imaging method of the present invention provides clear imaging and low cost, specifically in the following aspects:
[0075] This method enables fluorescent microbeads to be uniformly distributed in a monolayer on the gel surface, making it suitable for imaging with ordinary wide-field microscopes.
[0076] Patterning is an indirect transfer patterning scheme that avoids the stress deformation problem in soft lithography, while also avoiding damage to the gel surface caused by direct mechanical contact, thus improving process repeatability.
[0077] There are no requirements for lithography machines or photoresists, reducing equipment requirements. Deep ultraviolet lithography and special photosensitive gel materials are not needed.
[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A patterned traction force microscopic imaging method, characterized in that the steps include... include: a) Fabricating patterned templates on a hard substrate; b) Passivate the non-patterned areas of the patterned template, deposit adhesive proteins in the patterned areas to form a patterned adhesive protein layer, and apply a crosslinking agent to enhance the binding of the adhesive proteins to the gel substrate in step d). c) Deposit fluorescent microspheres on the surface of the adhesion protein layer to form a uniformly distributed layer of fluorescent microspheres; d) Prepare a gel substrate on the patterned template, and transfer the patterned adhesion protein layer and the fluorescent microsphere layer to the surface of the gel substrate to form a patterned gel substrate.
2. The patterned traction force microscopic imaging method according to claim 1, characterized in that, Step a) includes preparing the patterned template on the hard substrate using photolithography, specifically including: spin-coating photoresist, exposing through a mask, and developing to remove the photoresist from the non-patterned areas. The photoresist is a positive photoresist, and the spin-coating thickness is 1-2 μm.
3. The patterned traction force microscopic imaging method according to claim 1 or 2, characterized in that, The hard substrate is a circular cover glass sheet, which undergoes plasma cleaning and surface modification treatment before photolithography. The surface modification treatment includes hexamethyldisilazane vapor deposition.
4. The patterned traction force microscopic imaging method according to claim 1, characterized in that, In step b), the passivation treatment is performed using a passivating agent, which is a polylysine-polyethylene glycol copolymer with a concentration of 0.1–1 mg / mL; the adhesion protein is fibronectin with a concentration of 5–50 μg / mL; and the crosslinking agent is a 0.05% glutaraldehyde solution.
5. The patterned traction force microscopic imaging method according to claim 1, characterized in that, Step c) includes: mixing fluorescent microspheres with surfactants and solvents to form a solution, applying it to the surface of the patterned template, and forming the uniformly distributed fluorescent microsphere layer after the solvent evaporates.
6. The patterned traction force microscopic imaging method according to claim 5, characterized in that, The fluorescent microspheres have a solid content of 1% and a diameter of 0.2 μm; the surfactant is a 0.02% poloxamer solution; and the solvent is ethanol.
7. The patterned traction force microscopic imaging method according to claim 1, characterized in that, Step d) includes: covering the patterned template with a gel prepolymer solution, initiating polymerization to form the gel substrate, separating the gel substrate from the patterned template, and transferring the adhesion protein layer and the fluorescent microsphere layer to the surface of the gel substrate.
8. The patterned traction force microscopic imaging method according to claim 7, characterized in that, The gel prepolymer solution includes acrylamide and methylenebisacrylamide, with the concentration of acrylamide being 6% to 12% and the concentration of methylenebisacrylamide being 0.03% to 0.3%.
9. The patterned traction force microscopic imaging method according to claim 7, characterized in that, The gel substrate is attached to a silanized coverslip, the silanization process enhancing the adhesion between the gel substrate and the coverslip.