Methods for measuring cytodynamics in an in vitro fibrosis model

Abstract

A method for measuring cell dynamics in an in-vitro fibrosis model comprises: (a) providing a coaxial microscope system for imaging by assembling a plurality of microscope devices in a coaxial manner; (b) placing a sample on a stage and scanning it with the coaxial microscope system; (c) acquiring a first image and a second image of the sample using the coaxial microscope system, generating a third image from the first and second images, and contouring cell boundaries based on the third image; (d) defining a plurality of target regions within the third image; (e) identifying the mechanical properties of the sample, including calculating a first elastic modulus of cells and a second elastic modulus of proteins in the target region; and (f) comparing the first elastic modulus with the second elastic modulus to obtain the cellular state of the sample.

Description

Technical Field

[0001] Cross-reference of related applications This application claims the benefit of PCT Application No. PCT / CN2023 / 109368, filed on July 26, 2023. The entire disclosure of the above application is incorporated herein by reference.

[0002] The present invention relates to a method for measuring cell mechanics in an in vitro fibrosis model, and more specifically, to a method for simultaneously acquiring morphological images and mechanical properties of cells using a coaxial microscope system to evaluate cell states.

Background Art

[0003] As is well known in the art, conventional techniques for observing cells generally rely on fluorescence microscopy. This approach involves fluorescently labeling specific molecules or structures within a biological sample, after which the molecule or structure is excited by a selected light source to emit a detectable fluorescence signal. The high-contrast and high-resolution images generated in this manner enable an observer to visualize the molecular distribution and physiological processes within cells. Confocal microscopy, an improvement over fluorescence microscopy, employs a pinhole to focus the excitation beam onto a defined focal plane, while eliminating out-of-focus light, thereby improving image resolution and depth, reducing background noise, and enabling clearer three-dimensional imaging.

[0004] Atomic force microscopy (AFM) represents another approach for studying cells. AFM is a mechanical sensing microscopy technique that enables real-time visualization of the morphology and structure of the cell surface. By scanning the cell surface using a non-contact probe, AFM detects surface force interactions and generates detailed information about cell structure and organization. AFM also provides mechanical data such as surface topography, hardness, elastic modulus, and adhesion force, and can measure the mechanical properties of single biomolecules, making it useful for investigating cell membrane proteins, the extracellular matrix, and cell-cell interactions.

[0005] However, these existing technologies are not without limitations. Imaging still has to be performed separately, which prevents the simultaneous acquisition of complementary information. As a result, users have to spend additional time operating fluorescence microscopes and atomic force microscopes independently, verifying the accuracy of each scanning area, and securing sufficient laboratory space to accommodate both instruments.

[0006] Fibrosis is recognized as an irreversible pathological condition in humans. When tissue damage exceeds the body's repair capacity, the affected area transforms into fibrous tissue composed of fibroblasts and extracellular matrix proteins such as collagen and fibronectin. Increased tissue stiffness is considered a characteristic sign of organ fibrosis, and stimulation with transforming growth factor β (TGF-β) triggers activation of myofibroblasts, which correlates with the stiffness of the extracellular matrix. Currently, fibrous diseases such as keloids are mainly studied through cell-based experiments, and there is still no in vivo model suitable for investigating cell development across various stages of fibrosis.

[0007] In view of the above, the present invention provides a method for measuring cell dynamics in an in vitro fibrosis model that addresses the shortcomings of the prior art. [Overview of the Initiative]

[0008] The object of the present invention is to provide a method for measuring cell mechanics in an in vitro fibrosis model that enables effective observation and evaluation of the structural and mechanical properties of cells and extracellular proteins in a sample. The method employs a novel coaxial microscopy system that integrates a fluorescence microscope and an atomic force microscope in a coaxial configuration. By utilizing the technique of combining these two microscopes, users can simultaneously acquire detailed information on the morphological structure and mechanical properties of cells and extracellular proteins without having to perform separate operations. Thus, the method of the present invention provides a very convenient, efficient, and highly accurate approach for observing and evaluating cellular conditions.

[0009] To achieve the above objectives, the present invention discloses a method for measuring cell dynamics in an in vitro fibrosis model. The method comprises: (a) providing a plurality of microscope devices and assembling the microscope devices in a coaxial arrangement to constitute a coaxial microscope system for imaging a sample; (b) placing the sample on a stage and scanning the sample with the coaxial microscope system; (c) acquiring a first image and a second image of the sample with the coaxial microscope system, generating a third image from the first and second images, and contouring cell boundaries based on the third image; (d) defining a plurality of target regions for cells and proteins, respectively, within the third image; (e) identifying the mechanical properties of the sample, including calculating a first elastic modulus of cells and a second elastic modulus of proteins within the target region; and (f) acquiring the cellular state of the sample based on the third image by comparing the first elastic modulus with the second elastic modulus.

[0010] In one embodiment of the present invention, the target region has a variable shape, and the microscope device comprises a fluorescence microscope, an electron microscope, a laser scanning confocal microscope, a super-resolution fluorescence microscope, a scanning probe microscope, a magnetic tweezers microscope, an optical tweezers microscope, or an atomic force microscope.

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

[0012] In one embodiment of the present invention, the first image is a fluorescence image.

[0013] In one embodiment of the present invention, the first image is acquired from a laser scanning confocal microscope.

[0014] In one embodiment of the present invention, the second image is a height image, and the third image is a composite image.

[0015] In one embodiment of the present invention, the second image is obtained from an atomic force microscope.

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

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

[0018] In one embodiment of the present invention, the sample is a surviving unit of a fibrous disease.

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

[0020] In one embodiment of the present invention, fibrotic diseases include hepatic fibrosis, myelofibrosis, myocardial fibrosis, pulmonary fibrosis, cutaneous fibrosis, cancer-related fibrosis, or renal fibrosis.

[0021] In one embodiment of the present invention, the living cells are renal fibroblasts.

[0022] The detailed technology and preferred embodiments of the present invention will be described in the following paragraphs, together with the accompanying drawings, so that the problem, technical methods and embodiments of the claimed invention can be fully understood by those skilled in the art. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 is a flowchart showing a method for measuring cell mechanics in an in-vitro fibrosis model according to an embodiment of the present invention. [Figure 2] Figure 2 is a schematic diagram showing a simplified experimental workflow for selecting a target region and examining a sample using a coaxial integrated confocal microscope and an atomic force microscope according to an embodiment of the present invention. [Figure 3] Figure 3 shows representative fluorescence images (first image), morphological images (second image), and composite images (third image) of control samples and samples treated with transforming growth factor β1 (TGF-β1) for an embodiment of the present invention. [Figure 4] Figure 4 is a schematic diagram showing the fluorescein isothiocyanate (FITC) content in control samples and TGF-β1-treated samples according to an embodiment of the present invention. [Figure 5] Figure 5 is a schematic diagram showing a target region within a sample according to an embodiment of the present invention. [Figure 6] Figure 6 is a schematic diagram showing the elastic modulus of control samples and TGF-β1-treated samples according to an embodiment of the present invention. [Figure 7] Figure 7 is a schematic diagram showing the elastic modulus within the target region of control samples and TGF-β1-treated samples according to an embodiment of the present invention.

Embodiments for Carrying Out the Invention

[0024] Embodiments of the present invention are described in detail hereby with reference to the accompanying drawings. These embodiments are given for illustrative purposes only and are not intended to limit the present invention, its uses, or the specific embodiments described herein. Where applicable, the same reference numerals are used in the drawings and description to indicate the same or similar components. It should be noted that in the following embodiments and accompanying drawings, elements unrelated to the present invention have been omitted for simplicity, and the dimensional relationships between elements in the drawings are illustrated for clarity of understanding, not to represent actual scale.

[0025] Referring to Figure 1, the flowchart illustrates one embodiment of a method for measuring cell dynamics in an in-vitro fibrosis model according to the present invention. The basic steps include: (a) configuring a coaxial microscope system for imaging a sample by providing and assembling a plurality of microscope devices in a coaxial arrangement; (b) placing the sample on a stage and scanning it using the coaxial microscope system; (c) acquiring first and second images of the sample with the coaxial microscope system, generating a third image from the first and second images, and contouring cell boundaries based on the third image; (d) defining a plurality of regions of interest (ROIs) for cells and proteins, respectively, in the third image; (e) identifying the mechanical properties of the sample, which includes calculating a first elastic modulus of cells and a second elastic modulus of proteins within the region of interest; and (f) acquiring the cellular state of the sample based on the third image by comparing the first elastic modulus with the second elastic modulus.

[0026] Step (a) represents one of the key technical features of the method, namely the use of multiple microscopy devices. In this embodiment, two different types of microscopes are combined coaxially to constitute a coaxial microscopy system for imaging a sample. Microscopy devices may include, for example, a fluorescence microscope, an electron microscope, a laser scanning confocal microscope, a super-resolution fluorescence microscope, a scanning probe microscope, a magnetic force microscope, an optical force microscope, or an atomic force microscope (AFM). In particular, one microscope is used to acquire a fluorescence image of the sample, and the other is used to acquire an image that reflects the cell mechanics and structural morphology. In this embodiment, the combination of microscopy devices used in the method of the present invention comprises a laser scanning confocal microscope and an atomic force microscope, constituting a coaxial microscopy system of a laser scanning confocal microscope and an atomic force microscope. It should be noted that in this embodiment, the microscopy devices may be adapted according to the actual situation or specific usage requirements and are not limited to those described above.

[0027] In step (b), the sample is placed on the stage of a coaxial microscope system and scanned. In step (c), the coaxial microscope system acquires a first image and a second image of the sample, superimposes these two images to generate a third image, and outlines the cell boundaries based on the third image. Specifically, the first image is a fluorescence image of the sample, the second image is a surface height image of the sample, and the third image is generated by superimposing the first and second images. The first image is acquired from a microscope device capable of capturing fluorescence images, such as a laser scanning confocal microscope. The second image is acquired from a microscope device capable of capturing images of the mechanical properties and structural morphology of cells, such as an atomic force microscope. Next, step (d) involves defining multiple regions of interest (ROIs) with a variable shape that is inward or outward from the cell boundaries in the third image. In this embodiment, the cells and surrounding proteins can be clearly outlined as shown in Figure 3, and the region of interest is 1 μm. 2A square region is randomly or intentionally selected. It should be noted that in this embodiment, the number of pixels and size of the target region can be adjusted according to the actual situation or specific requirements, and are not limited to those described above.

[0028] After acquiring image data, step (e) is performed to identify the mechanical characteristics of the sample, such as rigidity, by calculating the first elastic modulus of cells and the second elastic modulus of proteins within the region of interest. Software analysis of the first and second elastic moduli provides mechanical information about both the cells and the surrounding proteins. The proteins in the region of interest may include elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers. In a preferred embodiment, the protein is a collagen fiber in a single structural form.

[0029] Step (f) involves using a first image acquired by a confocal (or fluorescence) microscope and a second image acquired by AFM to synthesize them into a third image in which the distribution, morphology, and mechanical structure of cells in the sample are accurately identified, and then the cell state is obtained by comparing the elastic modulus of the first and second images.

[0030] In this embodiment, the sample is a viable unit from a fibrous disease model, preferably a living cell such as renal fibroblasts. The fibrous disease may include hepatic fibrosis, myelofibrosis, myocardial fibrosis, pulmonary fibrosis, cutaneous fibrosis, cancer-associated fibrosis, or renal fibrosis. Preferred examples include renal fibrosis or keloid disease.

[0031] Materials and methods The method employs multiple fluorescent proteins for labeling to clearly distinguish cells from surrounding proteins. In this embodiment, rat renal fibroblasts (NRK-49F cells) were transfected to express Lifeact-RFP and cultured on a collagen gel conjugated with FITC-labeled type I collagen. The cultures were divided into a TGF-β1 treated group and a control group without TGF-β1. The addition of TGF-β1 simulated the activation of fibroblasts into myofibroblasts in fibrotic conditions such as renal fibrosis and keloids, allowing for analysis after cell contraction and collagen fiber interaction.

[0032] Coaxial AFM and Confocal Microscope In this embodiment, the coaxial microscope system combines a laser scanning confocal microscope (FV3000, Olympus) with an atomic force microscope (BioScope Resolve, Bruker) equipped with a sharp-tipped probe. Before measurement, the spring constant, deflection sensitivity, and probe position were calibrated using the AFM's no-touch mode and the confocal microscope's field of view.

[0033] Furthermore, prior to measurement, a hydrophobic material is coated onto the sharp tip of the atomic force microscope probe. This prevents the probe from adhering to collagen fibers during sample scanning, thereby maintaining measurement accuracy.

[0034] After calibration, the target area can be selected, imaged, and measured using a coaxial microscope system. The elastic modulus is calculated from the force-distance curve using an improved Sneddon model implemented in Bruker's data analysis software. Collagen fibers and cells are further analyzed for morphology and rigidity using NanoScope Analysis software (v1.9, Bruker).

[0035] Evaluation of cell-protein mechanical interactions Figure 2 schematically illustrates a simplified experimental workflow for selecting a target area and examining a sample using a coaxial microscopy system of laser scanning confocal microscopy and atomic force microscopy. A FITC-labeled collagen gel is prepared and seeded with Lifeact-RFP-transfected NRK-49F cells. After 24 hours of culture, a drug is added and the medium is replaced with fresh material. The prepared sample is mounted in the coaxial microscopy system, the scanning area is marked, and the sample is adjusted to the pre-marked area. Subsequently, images are acquired using the coaxial confocal and atomic force microscopy systems. Finally, fluorescence and morphological images are synthesized, and the mechanical properties of cells and collagen fibers are analyzed using software.

[0036] First, to distinguish the boundary between cells and fibers, an initial fluorescence image is acquired using a confocal microscope, as shown in Figure 3(A). Next, an initial height image is acquired using an atomic force microscope, as shown in Figure 3(B). Then, as shown in Figure 3(C), the cell boundary is identified based on the above. Finally, a square target area is selected, as shown in Figure 5. Analysis of the images acquired by the atomic force microscope shows, as shown in Figure 6, that treatment with TGF-β1 significantly increases the stiffness of collagen fibers and NRK-49F cells by approximately 6.5 and 6.7 times, respectively. Furthermore, as shown in Figure 7, it is observed that stiffer collagen fibers are frequently located near NRK-49F cells.

[0037] Next, a coaxial microscope system combining an atomic force microscope and a confocal microscope of the present invention is used to examine the morphology and mechanical properties of collagen fibers and living cells. The resulting images are shown in Figure 3. In the confocal microscope images, collagen fibers are clearly identifiable by FITC-labeled fluorescent protein, as shown in Figures 3(A1) and 3(A2), respectively, and NRK-49F cells are clearly identifiable by Lifeact-RFP fluorescent protein. When the two fluorescently labeled confocal images are combined, the relative positions of collagen fibers and NRK-49F cells within the same selected region can be confirmed, as shown in Figure 3(A3). Furthermore, as shown in Figures 3(B1) and 3(B2), the height images and 3D reconstructed images acquired by the atomic force microscope provide the morphology and mechanical properties of the cells and collagen fibers. To further confirm the accuracy of the coaxial microscope system, the confocal image and atomic force microscope image are combined and analyzed, as shown in Figure 3(C1). The combined image exhibits a high degree of overlap, facilitating a more precise analysis of the interaction between cells and collagen fibers in the selected region.

[0038] Furthermore, as shown in Figures 3 and 4, the density of collagen fibers significantly increases following TGF-β1 stimulation. As shown in Figure 5, atomic force microscopy images were further analyzed to measure the diameter of collagen fibers (D1), the angle between NRK-49F cells and collagen fibers (A), and the stiffness of collagen fibers and NRK-49F cells. Data analysis shows that there is no significant difference in the diameter of collagen fibers or the angle between collagen fibers and NRK-49F cells after TGF-β1 treatment (not shown). In addition, as shown in Figure 7, single collagen fibers (single structural morphology) were analyzed, and the change in stiffness was measured and normalized to the pericellular (CP) region. The results show that single collagen fibers located near NRK-49F cells (M) are stiffer than those located farther from NRK-49F cells (D).

[0039] These experimental results confirm that this method enables simultaneous observation of living cells and surrounding proteins in terms of distribution, morphological changes, and mechanical properties using a novel coaxial microscope system combining a laser scanning confocal microscope and an atomic force microscope. The coaxial microscope system of the present invention enables simultaneous acquisition of fluorescence and mechanical property images in a selected target region, and significantly reduces the time required to independently operate two microscope devices and align them to the same selected region. Furthermore, it reduces the space required to mount the two microscope devices, thereby enabling highly efficient and accurate identification of the cell state.

[0040] Although the present invention has been described in great detail with reference to its specific embodiments, other embodiments are also possible. Therefore, the spirit and scope of the appended claims should not be limited to the embodiments described herein.

Claims

1. A method for measuring cell dynamics in an in vitro fibrosis model, (a) Providing a plurality of microscope devices and assembling the microscope devices in a coaxial arrangement to constitute a coaxial microscope system for imaging a sample; (b) The steps of placing the sample on a stage and scanning the sample with the coaxial microscope system, (c) The steps of acquiring a first image and a second image of the sample using the coaxial microscope system, generating a third image from the first image and the second image, and outlining the cell boundaries based on the third image, (d) The step of defining multiple target regions in the third image with respect to cells and proteins, (e) A step of determining the mechanical properties of the sample, comprising the step of calculating a first elastic modulus of the cells and a second elastic modulus of the protein within the target region, (f) A step of obtaining the cellular state of the sample based on the third image by comparing the first elastic modulus with the second elastic modulus, A method for providing this.

2. The method according to claim 1, wherein the target region has a variable shape, and the microscope device comprises a fluorescence microscope, an electron microscope, a laser scanning confocal microscope, a super-resolution fluorescence microscope, a scanning probe microscope, a magnetic tweezers microscope, an optical tweezers microscope, or an atomic force microscope.

3. The method according to claim 2, wherein the microscope device is a combination of the laser scanning confocal microscope and the atomic force microscope.

4. The method according to claim 3, wherein the first image is a fluorescence image.

5. The method according to claim 4, wherein the first image is obtained from the laser scanning confocal microscope.

6. The method according to claim 3, wherein the second image is a height image and the third image is a composite image.

7. The method according to claim 6, wherein the second image is obtained from the atomic force microscope.

8. The method according to claim 1, wherein the protein comprises elastin fibers, fibronectin fibers, collagen fibers, or laminin fibers.

9. The method according to claim 8, wherein the protein is a collagen fiber having a single structural form.

10. The method according to claim 1, wherein the sample is a surviving unit of a fibrous disease.

11. The method according to claim 10, wherein the living unit is a living cell.

12. The method according to claim 10, wherein the fibrotic disease includes hepatic fibrosis, myelofibrosis, myocardial fibrosis, pulmonary fibrosis, cutaneous fibrosis, cancer-associated fibrosis, or renal fibrosis.

13. The method according to claim 11, wherein the living cells are renal fibroblasts.

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