Method for measuring cellular mechanics in in vitro fibrosis model

Abstract

The present invention relates to a method for measuring cellular mechanics in an in vitro fibrosis model, comprising: (a) providing a plurality of microscope devices, and combining same in a coaxial mode to form a microscope coaxial system for imaging; (b) placing on an object stage a sample to be measured, and scanning said sample by means of the microscope coaxial system; (c) capturing a first image and a second image of said sample by using the microscope coaxial system, generating the first image and the second image into a third image, and delineating a cell boundary by means of the third image; (d) defining a plurality of regions of interest in the third image; (e) determining the mechanical properties of said sample, comprising calculating a first elastic modulus of a cell and a second elastic modulus of a protein in the regions of interest; and (f) on the basis of the third image, comparing the first elastic modulus with the second elastic modulus to obtain the cell state of said sample.

Description

Methods for measuring cell mechanics in an in vitro fibrosis model Technical Field

[0001] The present invention relates to a method for measuring cell mechanics in an in vitro fibrosis model, and more particularly to a method for simultaneously collecting cell morphology images and mechanical properties using a coaxial microscope system to evaluate cell status. Background Art

[0002] Prior art methods for observing cells primarily rely on fluorescence microscopy, a microscopy technique that utilizes fluorescent labeling to observe biological samples. By labeling specific molecules or structures with fluorescent dyes and stimulating the labeled fluorescent dyes with a specific excitation light source, the fluorescent signal emitted by the dye is observed. This provides high-contrast and high-resolution imaging, enabling users to observe and study the molecular distribution and physiological processes of cells. Confocal microscopy, a further development of fluorescence microscopy, utilizes the confocal principle to focus the excitation light on the sample by passing the light beam through a narrow slit, while excluding scattered light from non-focal areas. This improves the resolution and depth of the image while reducing the impact of background noise, resulting in clearer and deeper three-dimensional images.

[0003] Another method for observing cells is to use atomic force microscopy, which is a microscopy technology based on mechanical sensing and can observe the morphology and structure of cell surfaces in real time. It involves using a non-contact fine needle probe to scan the cell surface. By detecting the force interaction on the surface, the cell's structure, texture and other morphological information can be obtained. On the other hand, atomic force microscopy can obtain the mechanical properties of cells, such as surface morphology, hardness, elastic modulus and adhesion. Atomic force microscopy technology can also measure the mechanical properties of single biomolecules, and can further be used to study the mechanical properties of cell membrane proteins, extracellular matrix and cell-to-cell interactions.

[0004] However, these technologies have limitations for cell observation. Currently, these techniques still require separate cell image capture and cannot be used together. Consequently, users must spend more time operating the fluorescence microscope and atomic force microscope independently, verifying the accuracy of the scanning area under independent operation, and requiring sufficient space to accommodate these two different microscope devices.

[0005] Fibrosis is an irreversible pathological condition in human disease. When normal organs or tissues are damaged beyond the body's ability to repair them, the damaged area transforms into fibrotic tissue composed of fibroblasts and an extracellular matrix containing collagen and fibronectin. Increased tissue stiffness is considered a pathological indicator of organ fibrosis, and stimulation with transforming growth factor-beta leads to myofibroblast activation, which is also correlated with extracellular matrix stiffness. Currently, fibrotic diseases such as keloid can only be studied through cell-based experiments, and suitable in vivo models are still lacking to study the cellular progression of fibrotic diseases at various stages.

[0006] In view of this, the present invention proposes a method for measuring cell mechanics in an in vitro fibrosis model to solve the problems encountered in the background art.

[0007] Summary of the Invention

[0008] The present invention aims to provide a method for measuring cell mechanics in an in vitro fibrosis model, aiming to effectively observe and evaluate the structure and mechanical properties of cells and extracellular proteins in the sample to be tested. The method of the present invention includes the use of a novel coaxial microscope system, specifically combining a fluorescence microscope and an atomic force microscope in a coaxial manner. By using the technology of the two coaxial microscope systems, users can simultaneously obtain detailed information on the morphological structure and mechanical properties of cells and extracellular proteins without the need for independent operation. Therefore, the method of the present invention provides a highly convenient, efficient and accurate way to observe and evaluate cell status.

[0009] To achieve the above-mentioned objectives, the present invention discloses a method for measuring cell mechanics in an in vitro fibrosis model, the method comprising: (a) providing a plurality of microscope devices, and coaxially combining the microscope devices into a microscope coaxial system to image the sample to be tested; (b) placing the sample to be tested on a stage, and scanning the sample to be tested via the microscope coaxial system; (c) capturing a first image and a second image of the sample to be tested using the microscope coaxial system, and then generating a third image from the first image and the second image, and dividing a cell boundary using the third image; (d) defining a plurality of regions of interest for cells and proteins in the third image, respectively; (e) confirming the mechanical properties of the sample to be tested, including calculating a first elastic modulus of the cells and a second elastic modulus of the proteins in the region of interest; and (f) obtaining the cell state in the sample to be tested based on the third image and comparing the first elastic modulus with the second elastic modulus.

[0010] In one embodiment, the region of interest is a non-fixed shape region of interest, and the microscope device includes a fluorescence microscope, an electron microscope, a laser scanning confocal microscope, an ultra-high resolution fluorescence microscope, a scanning probe microscope, a magnetic tweezers microscope, an optical tweezers microscope, or an atomic force microscope.

[0011] In one embodiment, the microscope device is a combination of a laser scanning confocal microscope and an atomic force microscope.

[0012] In one embodiment, the first image is a fluorescent image.

[0013] In one embodiment, the first image is captured from a laser scanning confocal microscope.

[0014] In one embodiment, the second image is a height image, and the third image is a fused image.

[0015] In one embodiment, the second image is captured from an atomic force microscope.

[0016] In one embodiment, the protein comprises elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers.

[0017] In one embodiment, the protein is a collagen fiber in a single structural form.

[0018] In one embodiment, the sample to be tested is a living body unit of a fibrotic disease.

[0019] In one embodiment, the living unit is a living cell.

[0020] In one embodiment, the fibrotic disease comprises liver fibrosis, myelofibrosis, cardiac fibrosis, lung fibrosis, skin fibrosis, cancer fibrosis, or kidney fibrosis.

[0021] In one embodiment, the living cell is a renal fibroblast.

[0022] After referring to the accompanying drawings and the embodiments described below, those skilled in the art will understand other objectives of the present invention, as well as the technical means and embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a flow chart of a method for measuring cell mechanics in an in vitro fibrosis model according to one embodiment of the present invention;

[0024] FIG2 is a schematic diagram of a simplified experimental process for selecting a region of interest and detecting a sample using a confocal microscope and an atomic force microscope coaxial system according to an embodiment of the present invention;

[0025] 3 is a fluorescence image (first image), a topographic image (second image), and a fusion image (third image) of a control group and a test sample treated with transforming growth factor-β1 according to an embodiment of the present invention;

[0026] 4 is a schematic diagram showing the fluorescein isothiocyanate (FITC) content in a control group of test samples and in a test sample treated with transforming growth factor-β1 (TGF-β1) according to an embodiment of the present invention;

[0027] FIG5 is a schematic diagram of a region of interest in a sample to be tested according to an embodiment of the present invention;

[0028] FIG6 is a schematic diagram of the elastic modulus of a control group of test samples and a sample treated with transforming growth factor-β1 (TGF-β1) according to an embodiment of the present invention; and

[0029] FIG. 7 is a schematic diagram of the elastic modulus of the region of interest of a control group of the test sample and a region of interest after being treated with transforming growth factor-β1 according to an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be explained below through examples. The examples of the present invention are not intended to limit the present invention to any specific environment, application, or special method as described in the examples. Therefore, the description of the examples is only for the purpose of illustrating the present invention and is not intended to limit the present invention. It should be noted that in the following examples and drawings, components not directly related to the present invention have been omitted and are not shown, and the dimensional relationships between the components in the drawings are only for ease of understanding and are not intended to limit the actual proportions.

[0031] Please refer to Figure 1, which shows a flow chart of the method for measuring cell mechanics in an in vitro fibrosis model provided by the present invention, the main steps of which are: step (a) providing multiple microscope devices, and combining the microscope devices in a coaxial manner into a microscope coaxial system to image the sample to be tested; (b) placing the sample to be tested on a stage, and scanning the sample to be tested through the microscope coaxial system; (c) capturing a first image and a second image of the sample to be tested using the microscope coaxial system, and then generating a third image from the first image and the second image, and dividing the cell boundaries using the third image; (d) defining multiple regions of interest for cells and proteins in the third image respectively; (e) confirming the mechanical properties of the sample to be tested, including calculating the first elastic modulus of the cell and the second elastic modulus of a protein in the region of interest; (f) obtaining the cell state in the sample to be tested based on the third image and comparing the first elastic modulus with the second elastic modulus.

[0032] Step (a) is one of the most important technical features of the method of the present invention, which includes the use of multiple microscope devices. Specifically, two different types of microscope devices are used in this embodiment, and the two different types of microscope devices are coaxially combined into a microscope coaxial system, and then the imaging of the sample to be tested is performed. Among them, the microscope device includes a fluorescence microscope, an electron microscope, a laser scanning confocal microscope, an ultra-high resolution fluorescence microscope, a scanning probe microscope, a magnetic tweezers microscope, an optical tweezers microscope, or an atomic force microscope, and the two different types of microscope devices are mainly divided into a microscope device for capturing fluorescence images of the sample to be tested, and a microscope device for capturing images of cell mechanical properties, structural appearance, etc. In this embodiment, the microscope device used in the method of the present invention is a combination of a laser scanning confocal microscope and an atomic force microscope, forming a microscope coaxial system of a laser scanning confocal microscope and an atomic force microscope. It should be noted that the microscope device in this embodiment can be adjusted according to the actual situation or usage requirements, and is not limited here.

[0033] The next step (b) involves placing the sample to be tested on the stage of a microscope coaxial system and scanning the sample using the microscope coaxial system. Step (c) involves using the microscope coaxial system to capture a first image and a second image of the sample to be tested. The first and second images are then superimposed to generate a third image, and the third image is used to delineate a cell boundary. Specifically, the first image is a fluorescence image of the sample to be tested, the second image is a surface height image of the sample to be tested, and the third image is an image generated by superimposing the first and second images. The first image is captured from a microscope capable of obtaining fluorescence images, such as a laser scanning confocal microscope. The second image is captured from a microscope capable of obtaining images of cell mechanical properties, structural features, and other features, such as an atomic force microscope. Next, step (d) involves defining multiple, non-fixed regions of interest (ROIs) in the third image, extending inward or outward from the cell boundary. In this embodiment, the cell and surrounding proteins can be clearly defined as shown in Figure 3. The ROIs are square, with one-square-micrometer squares selected randomly or non-randomly as the ROIs. It should be noted that the number of pixels and the region of interest in this embodiment can be adjusted according to actual scenarios or usage requirements and are not limited here.

[0034] After obtaining the image data in the above steps, step (e) can be performed. Confirming the mechanical properties of the sample, such as the rigidity of the sample, involves calculating a first elastic modulus for the cells in the sample and a second elastic modulus for the proteins in the region of interest. Software analysis of the first and second elastic moduli yields mechanical property data for the cells and surrounding proteins. Specifically, the proteins in the region of interest can include elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers. In preferred embodiments, the proteins are collagen fibers in a single structural form.

[0035] Finally, step (f) is performed. Based on the first image of the sample to be tested, i.e., the fluorescence image captured by the confocal microscope (or fluorescence microscope), and the second image of the sample to be tested, i.e., the height image captured by the atomic force microscope, the first image and the second image (i.e., the third image) are combined to accurately confirm the cell distribution, morphology, and mechanical property structure of the captured area in the sample to be tested, and the first elastic modulus of the cells and the second elastic modulus of the protein are compared to obtain the cell status in the sample to be tested.

[0036] In this embodiment, the sample to be tested is a living organism with a fibrotic disease, wherein the living organism is a living cell, and preferably, the living cell is a renal fibroblast. Furthermore, fibrotic diseases include liver fibrosis, myelofibrosis, cardiac fibrosis, pulmonary fibrosis, skin fibrosis, cancer fibrosis, or renal fibrosis, with renal fibrosis and keloid disease being preferred.

[0037] Materials and Methods

[0038] To clearly distinguish cells from surrounding proteins, the present method involves labeling with multiple different fluorescent proteins. In this example, rat kidney fibroblasts (NRK-49F cells) were transfected with the red fluorescent protein (Lifeact-RFP) polypeptide and cultured on a collagen gel containing type I collagen labeled with FITC and conjugated to the fluorescent protein. The cells were then divided into two groups: a group stimulated with transforming growth factor-β1 (TGF-β1) and a control group without TGF-β1. It should be noted that TGF-β1 was added to simulate the activation of other fibroblasts into myofibroblasts in fibrotic diseases (e.g., renal fibrosis and keloids). The interaction between cell contraction and collagen fibers will be further confirmed in the future.

[0039] [Coaxial atomic force microscope and confocal microscope]

[0040] The coaxial microscope system used in this example combines a laser scanning confocal microscope (FV3000, Olympus) and an atomic force microscope (BioScope Resolve, Bruker). This system also features a sharp probe. Prior to measurement using this coaxial microscope system, the probe's spring constant, deflection sensitivity, and probe position were calibrated using the AFM's non-contact mode within the confocal microscope's field of view.

[0041] In addition, before measurement, a hydrophobic substance must be coated onto the sharp probe of the atomic force microscope. This prevents the probe from sticking to the collagen fibers when scanning the sample to be measured, which would reduce the accuracy of the detection.

[0042] After calibration, regions of interest (ROIs) can be selected, captured, and measured using the coaxial system of the atomic force microscope and confocal microscope. The elastic modulus measured in this invention is calculated based on the measured force-distance curve using a modified Sneddon model using Bruker data analysis software. The morphology and stiffness of collagen fibers and cells are analyzed using software (NanoScope Analysis, v1.9, Bruker).

[0043] [Used to evaluate the mechanical interaction between cells and protein fibers]

[0044] Please also refer to Figure 2, which shows a schematic diagram of a simple experimental process for selecting an area of ​​interest and detecting a sample to be tested using a confocal microscope and an atomic force microscope coaxial system in the method for measuring cell mechanics in an in vitro fibrosis model provided by the present invention. The experimental process includes preparing a FITC-labeled collagen gel and then inoculating NRK-49F cells transfected with Lifeact-RFP. After 24 hours of cell culture, the specified drug is added and the culture medium is further updated. Next, the prepared sample is placed on the coaxial system of the microscope, the position of the scanning area is marked, and then the position of the selected sample is adjusted to the pre-marked area. Then, images are captured using a coaxial confocal microscope and an atomic force microscope. Finally, the fluorescence and morphology images are merged, and the mechanical properties of the cells and collagen are analyzed using software.

[0045] First, to distinguish the boundaries between cells and fibers, a raw fluorescence image was captured using a confocal microscope, as shown in Figure 3 (A). Next, a raw height image was obtained using an atomic force microscope, as shown in Figure 3 (B). The cell boundaries obtained above were then determined, as shown in Figure 3 (C). Finally, a square region of interest was selected, as shown in Figure 5. Data analysis of the AFM images revealed that the TGF-β1-treated group significantly increased the stiffness of collagen fibers and NRK-49F cells by 6.5-fold and 6.7-fold, respectively, as shown in Figure 6. Furthermore, stiffer collagen fibers were observed frequently in areas near NRK-49F cells, as shown in Figure 7.

[0046] Next, in order to detect the morphology and mechanical properties of collagen fibers and living cells, the coaxial system of the atomic force microscope and confocal microscope in the present invention is used. The image results obtained are shown in Figure 3. The confocal microscope image shows that collagen fibers can be clearly identified by the fluorescent protein FITC, and the fluorescent protein Lifeact-RFP can clearly identify NRK-49F cells, as shown in (A1) and (A2) in Figure 3, respectively. After the two fluorescently labeled confocal microscope images are fused, as shown in (A3) in Figure 3, the relative positions of collagen fibers and NRK-49F cells can also be confirmed in the same selected area. On the other hand, the morphology and mechanical properties of cells and collagen fibers can be obtained from the height image and three-dimensional remodeling image of the atomic force microscope image at the same time, as shown in (B1) and (B2) in Figure 3. To further confirm the accuracy of the coaxial system of the microscope, the confocal microscope image and the atomic force microscope image are fused and analyzed, as shown in (C1) in Figure 3. It was found that the fused images had a high degree of overlap, which was conducive to more accurate analysis of the interaction between cells and collagen fibers in the selected area.

[0047] In addition, as shown in Figures 3 and 4, the density of collagen fibers increased significantly after TGF-β1 stimulation. The diameter of the collagen fibers (D1), the angle between NRK-49F cells and the collagen fibers (A), and the hardness of the collagen fibers and NRK-49F cells were detected by atomic force microscopy, as shown in Figure 5. Next, data analysis showed that after TGF-β1 treatment, there was no difference in the diameter of the collagen fibers and the angle between the collagen fibers and the NRK-49F cells (not shown). In addition, single-structure collagen fibers can also be observed. As shown in Figure 7, the change factor on a single collagen fiber is measured and the value is normalized to the cell periphery (CP) area. The results show that the single-structure collagen fibers (M) close to the NRK-49F cells are harder than the single-structure collagen fibers (D) of the distal NRK-49F cells.

[0048] The above experimental results and data confirm that the method provided by the present invention for measuring cell mechanics in an in vitro fibrosis model includes the use of a novel microscope coaxial system, particularly a microscope coaxial system combining a laser scanning confocal microscope and an atomic force microscope, which can simultaneously observe the distribution, morphological changes, and mechanical properties of cells and proteins acting around the cells on a living unit. The microscope coaxial system of the present invention can simultaneously capture fluorescence images and mechanical property images in a selected area of ​​interest, greatly reducing the time required to independently operate two microscope devices and to align them with the same selected area, and can reduce the placement space required for the two microscope devices, thereby achieving the effect of obtaining cell status with high efficiency and high precision.

[0049] The above embodiments are merely examples of the present invention and illustrate the technical features of the present invention, and are not intended to limit the scope of protection of the present invention. Any modifications or equivalent arrangements that can be easily accomplished by those skilled in the art fall within the scope claimed by the present invention, and the scope of protection of the present invention shall be based on the claims.

Claims

1. A method for measuring cell mechanics in an in vitro fibrosis model, characterized in that: include: a) providing a plurality of microscope devices, and combining the microscope devices in a coaxial manner into a microscope coaxial system to perform imaging of a sample to be measured; b) placing the sample to be tested on the stage, and scanning the sample to be tested via the coaxial system of the microscope; c) capturing a first image and a second image of the sample to be tested by the coaxial system of the microscope, generating a third image by the first image and the second image, and dividing a cell boundary by the third image; d) defining a plurality of regions of interest for cells and proteins in the third image; e) confirming the mechanical properties of the sample to be tested, including calculating a first elastic modulus of the cells and a second elastic modulus of the protein in the region of interest; and f) According to the third image, the cell state in the sample to be tested is obtained by comparing the first elastic modulus with the second elastic modulus.

2. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 1, wherein: The region of interest is a non-fixed shape region of interest, and the microscope device includes a fluorescence microscope, an electron microscope, a laser scanning confocal microscope, an ultra-high resolution fluorescence microscope, a scanning probe microscope, a magnetic tweezers microscope, an optical tweezers microscope, or an atomic force microscope.

3. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 2, wherein: The microscope device is a combination of a laser scanning confocal microscope and an atomic force microscope.

4. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 3, wherein: The first image is a fluorescent image.

5. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 4, characterized in that: The first image is captured from a laser scanning confocal microscope.

6. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 3, wherein: The second image is a height image, and the third image is a fused image.

7. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 6, wherein: The second image is captured from an atomic force microscope.

8. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 1, wherein: The protein comprises elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers.

9. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 8, wherein: The protein is collagen fiber in the form of a single structure.

10. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 1, wherein: The sample to be tested is a living body unit of a fibrotic disease.

11. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 10, wherein: The living unit is a living cell.

12. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 10, wherein: The fibrotic disease includes liver fibrosis, bone marrow fibrosis, cardiac fibrosis, lung fibrosis, skin fibrosis, cancer fibrosis, or kidney fibrosis.

13. The method for measuring cell mechanics in an in vitro fibrosis model according to claim 11, wherein: The living cell is a kidney fibroblast.

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