TWI860001B - Method for measuring cellular mechanics in an in vitro fibrosis model - Google Patents

Abstract

A method for measuring cellular mechanics in an in vitro fibrosis model comprises : (a) providing a plurality of microscope devices and assembling in a coaxial manner to form a microscope coaxial system for imaging; (b) placing the sample on a stage and scanning through the microscope coaxial system; (c) capturing a first image and a second image of the sample using the microscope coaxial system, generating the first image and the second image into a third image, and delineating cell boundary based on the third image; (d) defining a plurality of regions of interest in the third image; (e) determining the mechanical properties in the sample, including calculating a first elastic modulus of the cells and a second elastic modulus of protein in the regions of interest; and (f) obtaining the cell status in the sample, and comparing the first elastic modulus with the second elastic modulus.

Description

Methods for measuring cell mechanics in an in vitro fibrosis model

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 microscope coaxial system to evaluate cell status.

Among the common techniques, the method of observing cells mainly relies on fluorescence microscopy. Fluorescence microscopy is a microscopy technique that uses fluorescent labeling to observe biological samples. By labeling specific molecules or structures with fluorescent dyes and using a specific excitation light source to excite the labeled fluorescent dyes, the fluorescent signals emitted by the dyes are observed. By providing high-contrast and high-resolution imaging, users can observe and study the molecular distribution and physiological processes of cells. Among them, the conjugate microscope is a technology further developed on the basis of the fluorescent microscope. It uses the conjugate focus principle to concentrate the excitation light on the focus of the sample by passing the light beam through a slit, while excluding the scattered light at the non-focal point. This can improve the resolution and depth of the imaging, while reducing the impact of background noise, and obtain clearer and deeper three-dimensional imaging.

Another way to observe cells is to use atomic force microscopy, which is a microscopic technology based on mechanical sensing. It can observe the morphology and structure of the cell surface in real time. It involves using a non-contact 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 morphological characteristics, hardness, elastic modulus and adhesion. And through atomic force microscopy technology, it is also possible to measure the mechanical properties of a single biological molecule, which can be further used to study the mechanical properties of cell membrane proteins, extracellular matrix and cell-to-cell interactions.

However, the above technologies have some limitations in cell observation. At present, these technologies still need to capture cell images separately and cannot be used together. Therefore, users need to spend more time to operate the fluorescent microscope and atomic force microscope independently, and confirm the accuracy of the scanned area under independent operation, and need enough space to place these two different microscope devices.

Fibrosis is known to be an irreversible pathological state in human diseases. When normal organs or tissues are damaged beyond the body's ability to repair, the damaged area will be transformed into a fibrotic tissue composed of fibroblasts and an extracellular matrix containing collagen and fibronectin. In addition, increased tissue hardness has been considered one of the pathological indicators of organ fibrosis, and the stimulation of type-B transforming growth factor will lead to the activation of myofibroblasts, which is also related to the hardness of the extracellular matrix. Currently, fibrotic diseases such as keloid can only be studied through cell experiments, and there is still a lack of suitable in vivo models to study the cell development process at various stages of fibrotic diseases.

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 prior art.

The purpose of the present invention is 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, especially combining a fluorescent microscope and an atomic force microscope in a coaxial manner. Through the technology of the two coaxial microscope systems, the user can simultaneously obtain detailed information on the morphological structure and mechanical properties of cells and extracellular proteins without the need for independent operations. Thus, the method of the present invention provides a highly convenient, efficient and accurate way to observe and evaluate cell status.

To achieve the above-mentioned object, 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 combining the microscope devices into a microscope coaxial system in a coaxial manner to image a sample to be tested; (b) placing the sample to be tested on a stage, and scanning the sample to be tested by the microscope coaxial system; (c) capturing a first image and a second image of the sample to be tested by the microscope coaxial system, and then imaging the first 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 proteins in the regions 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.

In one embodiment, the region of interest is a non-fixed shape region of interest, and the microscope device includes a fluorescent microscope, an electron microscope, a laser scanning conjugate microscope, an ultra-high resolution fluorescent microscope, a scanning probe microscope, a magnetic tweezer microscope, an optical tweezer microscope, or an atomic force microscope.

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

In one embodiment, the first image is a fluorescent image.

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

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

In one embodiment, the second image is captured by an atomic force microscope.

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

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

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

In one embodiment, the living unit is a living cell.

In one embodiment, the fibrotic disease comprises liver fibrosis, bone marrow fibrosis, heart fibrosis, lung fibrosis, skin fibrosis, cancer fibrosis, or kidney fibrosis.

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

After referring to the drawings and the implementation methods described subsequently, a person having ordinary knowledge in this technical field will understand other objects of the present invention, as well as the technical means and implementation modes of the present invention.

The content of the present invention will be explained below through embodiments. The embodiments of the present invention are not intended to limit the present invention to any specific environment, application or special method as described in the embodiments. Therefore, the description of the embodiments is only for the purpose of explaining the present invention, and is not intended to limit the present invention. It should be noted that in the following embodiments and drawings, components that are not directly related to the present invention have been omitted and not shown, and the size relationship between the components in the drawings is only for easy understanding and is not intended to limit the actual proportion.

Please refer to FIG. 1, which is a flow chart of the method for measuring cell mechanics in an in vitro fibrosis model provided by the present invention, and its main steps are: step (a) providing a plurality of microscope devices, and combining the microscope devices into a microscope coaxial system in a coaxial manner to image the sample to be tested; (b) placing the sample to be tested on a stage, and scanning the sample to be tested by the microscope coaxial system; (c) capturing a first image and a second image of the sample to be tested by the microscope coaxial system; (d) in the third image, defining a plurality of regions of interest for cells and proteins 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 a protein 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.

Step (a) is one of the most important technical features of the method of the present invention, and includes using a plurality of microscope devices. Specifically, in this embodiment, two different types of microscope devices are used, and the two different types of microscope devices are coaxially combined into a microscope coaxial system, and then imaging of the sample to be tested is performed. The microscope device includes a fluorescent microscope, an electron microscope, a laser scanning confocal microscope, an ultra-high resolution fluorescent microscope, a scanning probe microscope, a magnetic tweezer microscope, an optical tweezer microscope, or an atomic force microscope, etc., and the two different types of microscope devices are mainly divided into a microscope device for capturing fluorescent images of samples 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 concentric focus microscope and an atomic force microscope, forming a microscope coaxial system of a laser scanning concentric focus 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 use requirements, and is not limited here.

The next step (b) is to place the sample to be tested on the stage of the microscope coaxial system and scan the sample to be tested through the microscope coaxial system; step (c) is to use the microscope coaxial system to capture a first image and a second image of the sample to be tested, and then overlap the first image and the second image to generate a third image, and use the third image to delineate a cell boundary. Specifically, the first image is a fluorescent 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 overlapping the first image and the second image. The first image is captured from a microscope device that can obtain fluorescent images, such as a laser scanning concentric focus microscope. The second image is captured from a microscope device that can obtain images of cell mechanical properties, structural appearance, etc., such as an atomic force microscope. Then, step (d) is to define multiple and non-fixed ROIs from the cell boundary inward or outward in the third image. In this embodiment, the cell and the surrounding protein can be clearly defined by Figure 3, and the ROI is a square ROI, and a square area of one square micrometer is randomly or non-randomly selected as the ROI. It should be noted that the number of pixels and the ROI in this embodiment can be adjusted according to the actual situation or usage requirements, and are not limited here.

After obtaining the image information according to the above steps, the next step (e) can be performed. Confirming the mechanical properties of the sample to be tested, such as the rigidity of the sample to be tested, includes calculating a first elastic modulus of the cells in the sample to be tested and a second elastic modulus of the protein in the region of interest. After analyzing the first elastic modulus and the second elastic modulus with software, the mechanical property information of the cells and the surrounding proteins can be obtained respectively. Specifically, the protein in the region of interest can include elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers, wherein the protein of the preferred embodiment is a collagen fiber in a single structural form.

Finally, step (f) is performed. Based on the first image of the sample to be tested, i.e., the fluorescent image captured by the concentric focus microscope (or fluorescent 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 is compared with the second elastic modulus of the protein to obtain the cell state in the sample to be tested.

In this embodiment, the sample to be tested is a living body unit of a fibrotic disease, wherein the living body unit is a living body cell, and the living body cell of the preferred embodiment is a renal fibroblast. In addition, the fibrotic disease includes liver fibrosis, bone marrow fibrosis, heart fibrosis, lung fibrosis, skin fibrosis, cancer fibrosis, or kidney fibrosis, wherein the fibrotic disease of the preferred embodiment is renal fibrosis and keloid disease.

[Materials and methods]

In order to clearly distinguish cells from proteins that function around cells, the method of the present invention includes labeling with multiple different fluorescent proteins. In this example, rat kidney fibroblasts (hereinafter referred to as NRK-49F cells) were transfected and expressed with a polypeptide red fluorescent protein (hereinafter referred to as Lifeact-RFP), cultured on a collagen gel coupled to fluorescein isothiocyanate (hereinafter referred to as FITC) of the fluorescent protein, the collagen gel comprising type I collagen labeled with fluorescein isothiocyanate, and then divided into a group treated with transforming growth factor-β1 (hereinafter referred to as TGF-β1) and a control group (control) without TGF-β1. It should be noted that TGF-β1 was added here to simulate the process of activating myofibroblasts from other fibrotic cells in fibrotic diseases (such as renal fibrosis and keloid), and the interaction between cell contraction and collagen fibers will be further confirmed in the future.

[Coaxial atomic force microscope and concentric focus microscope]

The microscope coaxial system used in this embodiment is a combination of a laser scanning confocal microscope (FV3000, Olympus) and an atomic force microscope (BioScope Resolve, Bruker). This microscope coaxial system also has the use of a sharp probe. Before using this microscope coaxial system for measurement, the spring constant, deflection sensitivity and probe position of the probe are calibrated using the non-contact mode in the atomic force microscope and in the field of view of the confocal microscope.

In addition, before measurement, a hydrophobic substance must be coated onto the sharp probe of the atomic force microscope. This can prevent the probe from sticking to the collagen fibers when the atomic force microscope is scanning the sample to be tested, thereby reducing the accuracy of the detection.

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

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

Please refer to Figure 2, which is a schematic diagram of a simple experimental process provided by the present invention for measuring cell mechanics in an in vitro fibrosis model using a co-concentric microscope and an atomic force microscope co-axial system to select an area of interest and detect the sample to be tested. 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 microscope co-axial system, the position of the scanning area is marked, and then the position of the selected sample is adjusted to the pre-marked area. Next, images were captured using a coaxial confocal microscope and an atomic force microscope. Finally, the fluorescence and morphological images were merged and the mechanical properties of cells and collagen were analyzed using software.

First, in order to distinguish the boundary between cells and fibers, the original fluorescence image was captured using a confocal microscope, as shown in Figure 3 (A). Then, the original height image of the atomic force microscope was read, as shown in Figure 3 (B). Then, the cell boundary obtained as described above was shown in Figure 3 (C). Finally, a square region of interest was selected, as shown in Figure 5. After data analysis of the images captured by the atomic force microscope, it was shown that the group treated with TGF-β1 significantly increased the stiffness of collagen fibers and NRK-49F cells, by 6.5 times and 6.7 times, respectively, as shown in Figure 6. In addition, it can be observed that relatively stiff collagen fibers often appear in the area close to NRK-49F cells, as shown in FIG7 .

Next, in order to detect the morphology and mechanical properties of collagen fibers and living cells, the microscope coaxial system of the atomic force microscope and the coaxial microscope of the present invention was used. The image results obtained are shown in Figure 3. The coaxial microscope image shows that the collagen fibers can be clearly identified by the fluorescent protein FITC, and the NRK-49F cells can be clearly identified by the fluorescent protein Lifeact-RFP, as shown in Figure 3 (A1) and (A2), respectively. After the two fluorescently labeled coaxial microscope images are fused, as shown in Figure 3 (A3), the relative positions of the 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 reconstruction image of the atomic force microscope image, as shown in Figure 3 (B1) and (B2). To further confirm the accuracy of the microscope coaxial system, the concentric focus microscope image and the atomic force microscope image are fused and analyzed, as shown in Figure 3 (C1). It is found that the fused images have a high degree of overlap, which is conducive to a more accurate analysis of the interaction between cells and collagen fibers in the selected area.

In addition, as shown in Figures 3 and 4, the density of collagen fibers increased significantly after TGF-β1 stimulation. The diameter of collagen fibers (D1), the angle between NRK-49F cells and collagen fibers (A), and the hardness of collagen fibers and NRK-49F cells were detected by atomic force microscopy images, as shown in Figure 5. Next, data analysis showed that after TGF-β1 treatment, there was no difference in the diameter of collagen fibers and the angle between collagen fibers and NRK-49F cells (not shown). In addition, single-structure collagen fibers can also be observed. As shown in FIG7 , the multiple change on a single collagen fiber is measured and the value is normalized to the cell periphery (CP) region. The results show that the single-structure collagen fiber (M) close to the NRK-49F cell is harder than the single-structure collagen fiber (D) at the far end of the NRK-49F cell.

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, especially a microscope coaxial system combining a laser scanning co-focus microscope and an atomic force microscope, which can simultaneously observe the distribution and morphological changes of cells and proteins acting around cells in a living unit. The coaxial microscope system of the present invention can simultaneously capture the fluorescence image and the mechanical property image in a selected area of interest, greatly reducing the time required to independently operate two microscope devices and 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 the cell state with high efficiency and high accuracy.

The above embodiments are only used to illustrate the implementation of the present invention and to explain the technical features of the present invention, and are not used to limit the scope of protection of the present invention. Any changes or equivalent arrangements that can be easily completed by those familiar with this technology are within the scope of the present invention, and the scope of protection of the present invention shall be based on the scope of the patent application.

without.

Figure 1 is a flow chart of a method for measuring cell mechanics in an in vitro fibrosis model according to an embodiment of the present invention; Figure 2 is a schematic diagram of a simple experimental process for selecting an area of interest and detecting a sample to be tested using a coaxial microscope and an atomic force microscope coaxial system according to an embodiment of the present invention; Figure 3 is a fluorescent image (first image), a morphology image (second image) and a fusion image (third image) of a control group of a sample to be tested and treated with type-transforming growth factor-β1 (TGF-β1) according to an embodiment of the present invention; Figure 4 is a schematic diagram of the content of fluorescein isothiocyanate (FITC) in a control group of a sample to be tested and treated with type-transforming growth factor-β1 according to an embodiment of the present invention; FIG. 5 is a schematic diagram of a region of interest in a sample to be tested according to an embodiment of the present invention; FIG. 6 is a schematic diagram of elastic modulus of a control group of a sample to be tested according to an embodiment of the present invention and after treatment with type-beta transforming growth factor-(TGF-β1); and FIG. 7 is a schematic diagram of elastic modulus of a control group of a sample to be tested according to an embodiment of the present invention and after treatment with type-beta transforming growth factor-(TGF-β1).

Claims (9)

A method for measuring cell mechanics in an in vitro fibrosis model, the method comprising: (a) providing a plurality of microscope devices, and combining the microscope devices in a coaxial manner into a microscope coaxial system to image a sample to be tested, wherein the microscope device comprises a fluorescent microscope, an electron microscope, a laser scanning concentric focus microscope, an ultra-high resolution fluorescent microscope, a scanning probe, and a scanning probe. The invention relates to a method for preparing a sample to be tested, wherein the sample is a needle microscope, a magnetic tweezer microscope, an optical tweezer microscope, or an atomic force microscope, wherein the microscope device is a combination of a laser scanning concentric focus microscope and an atomic force microscope; (b) placing a sample to be tested on a stage, and scanning the sample to be tested by the microscope coaxial system; (c) capturing a first image and a second image of the sample to be tested by the microscope coaxial system, and then The first image and the second image are generated into a third image, and a cell boundary is delineated by the third image, wherein the first image is a fluorescent image, the second image is a height image, and the third image is a fused image; (d) a plurality of regions of interest are defined in the third image for cells and proteins by the microscope coaxial system, wherein the regions of interest are non (e) confirming the mechanical properties of the sample to be tested by the microscope coaxial system, including calculating a first elastic modulus of the cell and a second elastic modulus of the protein in the region of interest; and (f) obtaining the cell state in the sample to be tested by the microscope coaxial system based on the third image and comparing the first elastic modulus with the second elastic modulus. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 1, wherein the first image is captured from a laser scanning confocal microscope. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 1, wherein the second image is captured by an atomic force microscope. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 1, wherein the protein comprises elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 4, wherein the protein is a collagen fiber in a single structural form. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 1, wherein the sample to be tested is a living unit of a fibrosis disease. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 6, wherein the living unit is a living cell. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 6, wherein the fibrosis disease comprises liver fibrosis, bone marrow fibrosis, heart fibrosis, lung fibrosis, skin fibrosis, cancer fibrosis, or kidney fibrosis. A method for measuring cell mechanics in an in vitro fibrosis model as described in claim 7, wherein the living cell is a renal fibroblast.