CN117074405A - Method for measuring cell traction - Google Patents
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Abstract
The invention relates to a cell traction force measuring method, which comprises the steps of acquiring images of a substrate inoculated with cells to be measured according to a time sequence by using a phase-contrast microscope, wherein the images contain deformation information of the cells to be measured, acquiring a displacement field applied to the substrate by the cells to be measured according to the deformation information, acquiring a stress field applied to the substrate by the cells to be measured based on the displacement field, and acquiring a value of strain energy based on the displacement field and the stress field, wherein the value of the strain energy represents the cell traction force of the cells to be measured. The method for measuring the cell traction force uses white light as a light source, and performs label-free measurement on the cell traction force by means of distribution and refractive index difference of different structural components in the substrate, so that the requirements on microscope equipment are reduced, the sample preparation process is simplified, and the cell phototoxicity is reduced.
Description
Technical Field
The invention relates to the technical field of cell mechanics, in particular to a method for measuring cell traction.
Background
The whole life process of the organism is indistinguishable from the surrounding mechanical environment. The mechanical forces generated by the cells not only drive the bending, stretching, alignment and repositioning required for tissue development and homeostasis, but also regulate cellular functions from receptor signaling and transcription to differentiation and proliferation. The study of the mechanical characteristics and regularity of cell-cell and cell-extracellular matrix interactions depends on the accurate characterization of cell traction.
Common methods of cell traction measurement are: fluorescent molecular probe methods (fluorescence resonance energy transfer), micropillar methods (micropillr) and traction microscopy (Traction Force Microscopy, TFM).
Wherein, the fluorescent molecular probe method comprises a fluorescent acceptor and a fluorescent donor which are respectively arranged on the fluorescent acceptor and the fluorescent donor and are modified on a substrate, and a segment of elastic spiral deoxyribonucleic acid (DNA) fragment is connected between the fluorescent acceptor and the fluorescent donor in a covalent bond mode. When the cells adhere, the integrin molecules on the surface of the cell membrane are combined with the matrix modified by the fluorescence donor; the cell traction is transferred to the DNA fragment, so that the DNA fragment is stretched, and the distance between donor and acceptor molecules is increased; measuring the relative fluorescence intensity ratio of donor and acceptor molecules in a resting state and when the cell applies traction force through a fluorescence microscope, and estimating the variation of the distance between the donor and acceptor molecules; and (3) calculating the cell traction force by combining the elasticity coefficient of the DNA spring. The design and verification of fluorescent molecular probe experiments requires a lot of effort, and the relative fluorescence intensity ratio of donor and acceptor molecules may be changed by reasons other than the distance between the donor and acceptor molecules, such as degradation.
The microcolumn method calculates the traction of cells by inoculating the cells on an elastic microcolumn array according to the bending of the elastic microcolumn under the cells and combining the deflection of the microcolumn and the spring constant thereof. The microcolumn method simplifies calculation of traction applied by cells to a substrate, but is limited in that the cells must adhere to microcolumns having unique surface morphology, which may have an influence on the magnitude and distribution of traction of the cells.
The traction microscope technology is to inoculate cells on the surface of a substrate containing trace particles, record trace particle images of the surface of the substrate before and after cell adhesion or migration by using a confocal microscope, and calculate the displacement field of the substrate by combining an image processing algorithm; and then, calculating a cell stress field according to the Young modulus of the substrate and a displacement field inversion algorithm.
The above method has the following disadvantages:
1. trace particle aspect. Traction force microscopy based on tracer particles whose spatial resolution depends on the volume fraction of the tracer particles, makes it practically necessary to balance between high tracer particle density to achieve better spatial resolution and low tracer particle density to avoid endocytosis of phagocytic particles affecting normal physiological activities of cells and causing measurement errors.
Jacob Notbohm et al propose fluorescent protein labeling of substrate materials (quantifyingcell-induced matrix deformation in three dimensions based on imaging matrix fibers). The method needs to provide specially constructed fluorescent protein for each substrate material, has high technical difficulty and high sample preparation cost, and is difficult to eliminate the influence of the introduction of the fluorescent protein on the material. Jihan Kim et al propose to image the substrate topography by confocal microscopy in reflection mode, and to measure the cell traction (Three-Dimensional Reflectance Traction Microscopy). The method uses back-scattered light of microstructures in the material to form an image without requiring fluorescent labeling of the material. The method requires materials with high reflectivity, such as I-type protein, which limits the application range; meanwhile, when the microstructure in the sample is perpendicular to the direction of the light path, the problem that the microstructure is easy to miss in imaging exists due to the small reflection section.
2. And the economical aspect. The ultra-high resolution microscope represented by the confocal laser microscope has high equipment cost, and the time can reach nearly thousand yuan per hour.
3. Phototoxicity aspects. The laser confocal microscope or the fluorescence microscope has high phototoxicity to cells, especially sensitive cells such as nerve cells, and the activity of the cells is influenced by the illumination intensity and the illumination time, so that the traction and dynamics of the cells cannot be truly reflected, and the cells are possibly damaged. For example, experimental results show that the confocal microscope uses laser as a light source, the power per unit area is higher, the cell activity is reduced during imaging, and the measured cell traction is far lower than a true value.
4. Applicability is improved. The required spatial resolution of the traction microscope with the cell scale or larger subcellular scale is 1-10 microns, the resolution of the traditional bright field and fluorescent microscope is enough to meet the requirement, and an ultra-high resolution microscope is not needed.
Therefore, the traditional cell traction force measurement has the problems of high equipment requirement, complicated sample preparation, high cytotoxicity and the like.
Disclosure of Invention
Therefore, the invention aims to overcome the defects of the prior art and provide a cell traction force measuring method, which simplifies the sample preparation of a traction force microscope, reduces the use threshold of the cell traction force measuring method, avoids the use of laser/fluorescence, reduces cytotoxicity, more accurately characterizes the mechanical characteristics of cells, and can be widely applied to the technical fields of cell force measurement and the like.
According to a first aspect of the present invention, there is provided a method of measuring cell traction, comprising: acquiring images of a substrate inoculated with cells to be detected according to a time sequence by using a phase-contrast microscope, wherein the images contain deformation information of the cells to be detected; acquiring a displacement field applied to the substrate by the cell to be detected according to the deformation information; based on the displacement field, obtaining a stress field applied to the substrate by the cell to be detected; and obtaining a value of strain energy based on the displacement field and the stress field, wherein the value of strain energy characterizes a cell traction force of the cell under test.
Preferably, the substrate is a protein hydrogel substrate.
Preferably, the protein hydrogel substrate has a thickness of 50-1000 microns; the Young's modulus of the hydrogel substrate is 10-1000Pa.
Preferably, the protein hydrogel substrate has a protein concentration of 1-20 milligrams per milliliter.
Preferably, the acquiring the displacement field includes: acquiring images of the substrate at different moments; calculating the instantaneous displacement fields of the substrate at different moments; summing the instantaneous displacement fields at different moments to obtain an accumulated deformation field; and obtaining a displacement field applied to the substrate by the cell to be detected according to the accumulated deformation field.
Preferably, calculating the instantaneous displacement field of the substrate at different moments comprises: obtaining the similarity of the images of the substrate at different moments through a cross-correlation function; and acquiring the instantaneous displacement fields of the substrate at different moments according to the similarity.
Preferably, acquiring the displacement field applied by the test cell to the substrate includes:
step 1, taking the base image at the initial moment as a reference image, and taking the base image at the later moment as a deformed image;
step 2, dividing the reference image into a plurality of sub-images F1 with the same size as a query window, and searching a deformed sub-image F2 which is most similar to the reference sub-image F1 in the deformed image;
step 3, determining the similarity between the reference sub-image F1 and the deformed sub-image F2 through a cross-correlation function, and acquiring an instantaneous displacement field between the reference sub-image F1 and the deformed sub-image F2 according to the similarity; wherein the cross-correlation function is:
wherein F is 1 (x, y) is the image gray value of the sub-image F1 at the (x, y) coordinates; f (F) 2 (x+u dx ,y+u dy ) To have (u) with the sub-image F1 dx ,u dy ) The distorted sub-image F2 of the transient displacement field has gray values at the (x, y) coordinates; and
and 4, executing steps 2 and 3 on all the reference sub-images F1 through a grading algorithm, and obtaining a displacement field applied to the substrate by the cells to be detected.
Preferably, the grading algorithm is used to increase the calculation speed and the spatial resolution of the displacement field, and comprises: the first sub-image F1 has a size of 64×64 pixels, the second sub-image F1 has a size of 32×32 pixels, and the third sub-image F1 has a size of 16×16 pixels.
Preferably, the acquiring the stress field comprises:
based on the displacement field u (r), by a two-dimensional fast fourier transform FT 2 Function calculation of frequency domain spatial displacement fieldWherein (1)>u (r) is the displacement field;
based on the frequency domain spatial displacement fieldBinding green's function->Calculating the spatial stress field of the frequency domain->Wherein->And
by inverse two-dimensional Fourier transform FT 2 -1 A real domain spatial stress field is calculated, wherein, t (r) is the stress field applied by the test cell to the substrate.
Preferably, the obtaining the value of the strain energy includes: calculating the value of strain energy applied to the substrate by the cell to be tested through a strain energy algorithm, wherein the formula of the strain energy algorithm is as follows:
wherein,and->Respectively a stress vector and a displacement vector, A p The pixel area, U, is the value of strain energy.
Compared with the prior art, the invention has the advantages that: 1. the equipment requirement of cell traction measurement is reduced; 2. the preparation of the sample of the substrate inoculated with the cells is simplified; 3. cytotoxicity is reduced.
Drawings
Embodiments of the invention are further described below with reference to the accompanying drawings, in which:
FIG. 1 is a flow chart of a method for measuring cell traction according to an embodiment of the invention;
FIG. 2 is a schematic diagram showing the interference between two light waves in the prior art;
FIG. 3 is a comparison of a prior art phase contrast microscope with a generic wide field microscope taken of a protein hydrogel substrate topography;
FIG. 4A is a graph of the morphology of the cell and protein substrate at the time of initial cell seeding according to one embodiment of the present invention;
FIG. 4B is a graph of cell and protein substrate morphology at 4 hours of cell seeding provided in accordance with one embodiment of the present invention;
FIG. 4C is a displacement field applied to a protein substrate by cells at 4 hours of cell seeding in accordance with one embodiment of the present invention;
FIG. 4D is a stress field applied to a protein substrate by cells at 4 hours of cell seeding in accordance with one embodiment of the present invention.
FIG. 5A is a graph of the morphology of a protein substrate at the time of initial seeding of cells according to the prior art in contrast to an embodiment of the present invention;
FIG. 5B is a graph of the morphology of a protein substrate at 4 hours of cell seeding in comparison to an embodiment of the present invention, according to the prior art;
FIG. 5C is a displacement field applied to a protein substrate by cells at 4 hours of cell seeding in contrast to an embodiment of the present invention, according to the prior art;
FIG. 5D is a stress field applied to a protein substrate by cells at 4 hours of cell seeding in contrast to an embodiment of the present invention, according to the prior art; and
fig. 6 is a phase contrast microscope imaging light path diagram according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail by means of specific examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
As described in the background art, the traditional cell traction force measurement has the problems of high equipment requirement, complicated sample preparation and high cytotoxicity.
In order to solve the above problems, the present invention provides a cell traction measuring method. The cell traction force measuring method takes white light as a light source, and carries out label-free measurement on the cell traction force by means of rich information provided by distribution of different structural components and refractive index differences in the substrate. The invention improves the traditional traction force microscope method in three aspects of reducing equipment requirements, simplifying sample preparation process and reducing cytotoxicity, reduces the use threshold of the traction force microscope on the premise of ensuring the effective contrast of images, and expands the application range of the traction force microscope.
The present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 is a flow chart of a method for measuring cell traction according to an embodiment of the invention. As shown in fig. 1, the method for measuring cell traction provided by the invention comprises the following steps:
acquiring images of the substrate inoculated with the cells to be detected according to a time sequence by using a phase-contrast microscope, wherein the images contain deformation information of the cells to be detected;
acquiring a displacement field applied to the substrate by the cell to be detected according to the deformation information;
based on the displacement field, obtaining a stress field applied to the substrate by the cell to be detected; and
and acquiring a value of strain energy by adopting a strain energy algorithm based on the displacement field and the stress field, wherein the value of strain energy represents the cell traction force of the cell to be tested.
Specifically, placing a hydrogel substrate inoculated with cells to be detected on a microscope objective table, wherein the shape of the hydrogel substrate deforms under the action of the cells to be detected; the hydrogel morphology is collected by a high-speed camera of a phase-contrast microscope; according to the shapes of the hydrogel substrates before and after deformation, the displacement field, stress field and strain energy applied to the substrates by the cells are calculated through a computer software program. Wherein the displacement field may be referred to as a deformation field.
The substrate material does not need to introduce fluorescent microspheres or carry out fluorescent protein marking, and the stress field is calculated by utilizing the structural information of the material. The complexity of sample preparation is greatly reduced.
The calculation method of the substrate displacement field comprises the following steps:
for the acquired protein substrate topography maps at different moments, taking the previous moment topography map as a reference image and the later moment topography map as a deformation image, calculating a displacement field (instantaneous displacement field) of the protein substrate at the moment by adopting a particle image velocimetry (Particle Image Velocimetry, PIV; matlab kit PIVlab). By summing the instantaneous displacement fields of the protein substrate at different times, the displacement fields (accumulated displacement fields) of the protein substrate at different times compared with the initial time are obtained. The specific calculation method is as follows:
1) Selecting a proper sub-image F1 from the reference image as a query window, and searching a sub-image F2 which is most similar to the query window near a corresponding area in the deformed image; wherein the size of the selectable window for the first query is 128×128 or 64×64; the second time 64 x 64 or 32 x 32 and the third time 32 x 32 or 16 x 16, the values chosen are the preferred dimensions obtained by experimentation.
2) The similarity of the two sub-images is determined by a cross-correlation function, and the displacement between the two sub-images is the displacement of the query window;
the cross-correlation function is:
wherein F is 1 (x, y) is the image gray value of the sub-image F1 at the (x, y) coordinates; f (F) 2 (x+u dx ,y+u dy ) To have (u) with the sub-image F1 dx ,u dy ) The distorted sub-image F2 of the transient displacement field has gray values at the (x, y) coordinates;
3) Repeating the processes 1 and 2 for all the query windows in the reference graph to obtain an integrated accumulated displacement field of the substrate;
4) Through a grading algorithm and the size of a query window is reduced, the calculation speed and the spatial resolution of the substrate displacement field are improved.
The calculation method of stress field applied by the cells to the substrate is as follows:
based on the obtained real-domain displacement field u (r) of the substrate, the frequency domain space displacement field can be calculated through Matlab function fft2Wherein->Wherein, the fft2 function is a function of Matlab software for two-dimensional fast Fourier transform, which returns the transformed result. Based on the frequency domain spatial displacement field->Binding green's function->Calculating the spatial stress field of the frequency domain->Wherein the method comprises the steps of
Wherein the displacement field is composed of stress fieldAnd Green's function->Is given by the convolution of (a). Wherein (1)>To map the stress field to the green's function of the displacement field. In the Fourier domain space, < +.>From this, it can be seen that the frequency domain spatial stress field +.>
Based on the frequency domain spatial stress fieldThe real domain spatial stress field T (r) can be calculated in Mat lab software by using ifft2 function, wherein +_>The ifft2 function being an inverse two-dimensional Fourier transformIn addition, in addition to performing the calculation in Matlab software, the calculation solution may also be run in a program such as C++ or other software.
Furthermore, instead of using a fourier transform algorithm, the stress field applied by the test cells to the substrate can also be obtained using the DW method (also known as boundary element method, boundary Element Method, BEM). The DW method divides the contact area of cells and the elastic substrate into grids, and the corresponding cell traction force field is obtained by inversion of a displacement field under the assumption that the cell traction force acts on nodes of the grids in the form of concentrated force.
The calculation method for applying strain energy to the substrate by the cells is as follows:
the contractile force of a cell can be characterized by strain energy. For an elastic substrate, strain energy is the energy stored elastically in the substrate.
The formula for calculating strain energy:
wherein,and->Respectively a stress vector and a displacement vector, A p The pixel area, U, is the value of strain energy.
According to one embodiment of the invention, the displacement and stress fields are applied to the substrate by the cells shown in FIG. 4 according to the strain energy equation, combined with the Young's modulus of the substrate of 14.89 Pa, to give a strain energy applied to the substrate by the cells of 0.0295 picojoules.
And calculating the stress field applied by the cells to the substrate by combining the measured substrate displacement field with the Young modulus of the substrate and a Fourier frequency domain inversion algorithm. And then, calculating the value of strain energy applied by the cells to the matrix based on the substrate displacement field and the stress field, and facilitating quantitative characterization and comparison of the cell traction value.
The phase-contrast microscope converts the phase difference into the amplitude difference through the interference effect of light on the basis of a common optical microscope, so that the tiny difference of the thickness in the sample is converted into the light-dark difference, the contrast of the image is enhanced, and the sample is a hydrogel substrate inoculated with cells. The substrate represented by hydrogel has abundant structural information in different component distribution and refractive index difference, the structural information of the substrate is converted into the light-dark difference of an image through a phase-contrast microscope, and a high-contrast substrate topography diagram for substrate displacement field analysis can be obtained without modifying the substrate with fluorescent groups or introducing trace particles.
The invention selects an inverted phase-contrast microscope to replace the laser confocal of the traditional traction microscope method for image acquisition. The inverted phase-contrast microscope has low cost (1-10 ten thousand yuan/table), and only hundred yuan is needed per hour, so that the cost is better than that of a laser confocal microscope. The phase-contrast microscope resolution is determined by the diffraction theorem (resolution x, y=0.6λ/NA), where λ is the wavelength and NA is the numerical aperture of the phase-contrast microscope of the present invention. Taking magnification of 20×, numerical aperture NA of 0.45 and light source wavelength of 552 nm as an example, the spatial resolution is 736 nm, which satisfies the use requirement of traction microscope with cell scale or larger subcellular scale. Meanwhile, compared with wide-field imaging, the phase-difference microscope improves the contrast of images through phase-difference imaging, and ensures the accuracy of displacement field calculation.
The numerical aperture of the objective lens of the phase-contrast microscope is 0.3-0.95, and the magnification is 10-60 times; which can retard the direct or diffracted light by 1/4 lambda to 1/2 lambda.
The direct light and the diffracted light form a phase difference image on an image plane axis, the contrast of the image is enhanced, the imaging resolution of a high-precision substrate microstructure is provided, a time sequence micrograph is acquired by using a high-speed camera, a displacement field with high time and spatial resolution is obtained, and the measurement of a non-marking traction microscope is realized.
Fig. 2 is a schematic diagram showing the mutual interference between two light waves in the prior art, in which the change of the visible light wavelength and the frequency is represented by the difference of colors, the change of the amplitude is represented by the difference of brightness and darkness, and the change of the phase is not perceived by human eyes. When two light beams pass through the optical system, mutual interference occurs (when the two light beams are identical, the light amplitude is increased, the brightness is enhanced, otherwise, the two light beams are mutually offset, and the brightness is darkened). As shown in FIG. 2, the light is refracted after passing through the sample, deviates from the original light path, and is delayed by 1/4λ (wavelength), if the light path difference is changed to 1/2λ by increasing or decreasing by 1/4λ, the interference of the two beams after combining the axes is enhanced, the amplitude is increased or decreased, and the contrast is improved.
Fig. 3 is a comparison of a prior art phase contrast microscope with a generic wide field microscope taken profile of a protein hydrogel substrate. As shown in fig. 3, a Phase Contrast microscope (Phase Contrast) and a Wide Field microscope (Wide Field) are shown, wherein the Phase Contrast microscope has higher Contrast and more obvious brightness difference compared with the Wide Field microscope, so that the accuracy of displacement Field calculation is ensured, and meanwhile, the imaging mode of the Phase Contrast microscope has low illumination power per unit area and has small influence on the activity of cells.
FIG. 4A is a graph of the morphology of the cell and protein substrate at the time of initial cell seeding according to one embodiment of the present invention; FIG. 4B is a graph of cell and protein substrate morphology at 4 hours of cell seeding provided in accordance with one embodiment of the present invention; FIG. 5A is a graph of the morphology of a protein substrate at the time of initial seeding of cells according to the prior art in contrast to an embodiment of the present invention; FIG. 5B is a graph of the morphology of a protein substrate at 4 hours of cell seeding in comparison to an embodiment of the present invention, according to the prior art; as can be seen by comparing the image of FIG. 4B with the image of FIG. 5B, the contrast of the image of the substrate topography acquired by the phase contrast microscope is more obvious, the contrast of the image is higher, and the activity of the cell is also higher, thus ensuring the accuracy of the displacement field calculation.
FIG. 4C is a displacement field applied to a protein substrate by cells at 4 hours of cell seeding in accordance with one embodiment of the present invention; FIG. 4D is a stress field applied to a protein substrate by cells at 4 hours of cell seeding in accordance with one embodiment of the present invention. FIG. 5C is a displacement field applied to a protein hydrogel substrate by cells at 4 hours of cell seeding in contrast to an embodiment of the invention, according to the prior art; FIG. 5D is a stress field applied to a protein substrate by cells at 4 hours of cell seeding in contrast to an embodiment of the invention, according to the prior art.
According to one embodiment of the present invention, the young's modulus in fig. 4C is 20 pa, and as can be seen by comparing fig. 4C with fig. 5C, the size and the interval of the arrows in fig. 4C are more uniformly distributed, wherein the young's modulus of the protein hydrogel substrate in fig. 4C is selected within a range of 10-1000pa, the young's modulus is used for describing the mechanical properties of the substrate material, and the young's modulus value in a proper range is selected, so that the substrate can better sense the mechanical change of the cells, and the arrow size and the interval are more clearly and uniformly distributed on the image, which is more favorable for the accurate calculation of the displacement field. If the value of Young's modulus is selected too large, deformation of the substrate is not easily seen even if the cells are changed.
According to one embodiment of the present invention, fig. 4D shows stress fields calculated according to young's modulus values of the displacement field and the base material, wherein young's modulus is 20 pa, stress field is a force distribution, and is a depiction of force of the displacement field, and as can be seen by comparing fig. 4D with fig. 5D, the mechanical depiction in fig. 4D is more clear and accurate.
FIG. 6 is a view of an imaging light path of a phase contrast microscope according to the present invention, as shown in FIG. 6, a hydrogel substrate is placed on a stage of the phase contrast microscope, so that transmitted light 602 from a transmitted light source 601 can penetrate the hydrogel to reach a high-speed camera; the transmitted light 602 passes through an annular diaphragm 603 and a condenser 604 to form a hollow light cone 605 which is focused on the hydrogel substrate; the transmitted light passes through the sample 606, forming direct light 607 that passes directly through the sample and diffracted light 608 that avoids the sample; the direct light and the diffracted light diverge at the back focal plane of the objective lens 609, the direct light forming a light ring 610, the diffracted light 608 diverging outside the light ring; the phase plate 611 is located on the back focal plane of the objective lens 609, and the phase plate 611 is divided into a ring-shaped conjugate zone and a combining zone on both sides of the ring, the direct light passes through the conjugate zone, and the diffracted light passes through the combining zone; the conjugate or conjugate region of the phase plate is coated with a phase-retarding substance (usually magnesium fluoride) to retard the phase of light passing through the region; reference numeral 612 denotes a condenser, and the direct light and the diffracted light are coaxial in an image plane 613 to form a phase difference image, thereby enhancing the image contrast. Wherein the sample is a substrate inoculated with the test cells.
According to one embodiment of the invention, the magnification of the objective lens of the phase-contrast microscope of the invention is 20x, the numerical aperture NA is 0.45, the pixel size is 322 nm, the field size is 659 μm, the spatial resolution is 736 nm, the light source is 552 nm, and the resolution x, y=0.6λ/NA.
According to one embodiment of the invention, the seeding density of the test cells is: 1X 10 3 Individual cells/samples.
According to one embodiment of the invention, the phase-contrast microscope selected in the invention is a Nikon inverted phase-contrast microscope, and the visible light can be white light through visible light excitation, and the exposure time of the camera is 1 second. White light is used as a light source, and a matrix topography is obtained through the interference effect of light. Photodamage (phototoxicity) is weak, and the method is more suitable for traction measurement of sensitive cells and high-frame-rate image acquisition. If the laser is used as a light source, the fluorescent microsphere is excited. Cells are also irradiated by laser, and the photodamage (phototoxicity) is strong, which is unfavorable for the traction measurement of sensitive cells.
When the cells to be detected are initially inoculated, collecting an original morphology graph of the protein hydrogel substrate; topography of the protein hydrogel substrate was then acquired according to experimental requirements, for example, every 30 minutes.
After the cells to be tested are placed on the protein hydrogel matrix, the protein hydrogel matrix supporting the cells to be tested is deformed; the phase difference morphology images in the deformation process of the protein hydrogel matrix are acquired by a microscope camera according to the time sequence; the displacement field and the stress field applied by the cell to be detected to the protein hydrogel substrate are obtained by calculating the shape images of the hydrogel matrix before and after deformation by a computer software program (Matlab or other).
According to one embodiment of the invention, in order to better measure the cell traction force of the cell to be tested, the cell to be tested can generate enough deformation, and the thickness of the protein hydrogel substrate is 50-1000 microns; wherein, the range of the interval can be within (the vast majority of use scenes are covered); the selection of the thickness values during the experiment was dependent on the preparation of the samples. Hydrogel substrates with a thickness of less than 50 microns are not easy to prepare because the cells themselves have a thickness/height of about 10 microns and are not suitable for too thin a thickness. If it is thicker than 1000 microns, for an inverted microscope, the working distance of the vast majority of lenses is exceeded, i.e. the focal plane is in the substrate below the area where the cells are located (not in the beat).
According to one embodiment of the invention, the Young's modulus of the hydrogel substrate is 10-1000Pa. Wherein, the Young's modulus of the hydrogel is mainly influenced by the selection and preparation of materials, and 10-1000Pa covers the situation of most materials; excessive hardness, insignificant deformation of the substrate; too small, gravity causes significant deformation (affecting experimental results) when cells are seeded.
According to one embodiment of the invention, the protein hydrogel substrate has a protein concentration of 1-20 milligrams per milliliter.
The preparation method of the protein hydrogel substrate comprises the following steps:
(1) A2 mg/ml protein solution (for example, 200. Mu.l of 2 mg/ml protein solution was prepared by taking 10.57 mg/ml protein mother liquor as an example, 37.8. Mu.l of 10.57 mg/ml protein mother liquor, 0.87. Mu.l of 1 mol/l sodium hydroxide solution, 20. Mu.l of 10 Xphosphate buffer and 141.3. Mu.l of ultrapure water were required) was prepared on the ice box, and the components of the protein solution were uniformly mixed by blowing.
(2) 200. Mu.l of a 2 mg/ml protein solution was added to a glass bottom confocal cell culture dish (bottom slide diameter 14 mm) with a diameter of 29 mm, and the dish was allowed to solidify at 37℃for 30 minutes.
(3) After the protein is coagulated, adding 1x phosphate buffer solution to soak the protein hydrogel substrate, and keeping the protein hydrogel substrate for standby.
The invention is suitable for measuring the traction of cells on various substrate materials, for example, besides the protein hydrogel substrate used in the invention, proteins (Collagen), gelatin (Gelatin), matrigel (Matrigel), polyacrylamide (PAAM), polydimethylsiloxane (PDMS), methacrylic anhydride Gelatin (GelMA) and the like can be selected as substrate materials.
In addition, the invention is suitable for measuring cell traction on various vessel substrates, such as pore plates, organ chips, culture dishes and the like.
It should be noted that, although the steps are described above in a specific order, it is not meant to necessarily be performed in the specific order, and in fact, some of the steps may be performed concurrently or even in a changed order, as long as the required functions are achieved.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the technical improvements in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.
Claims (10)
1. A method of measuring cell traction, comprising:
acquiring images of a substrate inoculated with cells to be detected according to a time sequence by using a phase-contrast microscope, wherein the images contain deformation information of the cells to be detected;
acquiring a displacement field applied to the substrate by the cell to be detected according to the deformation information;
based on the displacement field, obtaining a stress field applied to the substrate by the cell to be detected; and
and acquiring a value of strain energy based on the displacement field and the stress field, wherein the value of strain energy characterizes the cell traction of the cell to be tested.
2. The method of cell traction measurement according to claim 1, wherein the substrate is a protein hydrogel substrate.
3. The cell traction measurement method according to claim 2, wherein the protein hydrogel substrate has a thickness of 50-1000 microns; the Young's modulus of the hydrogel substrate is 10-1000Pa.
4. The method of cell traction measurement according to claim 2, wherein the protein hydrogel substrate has a protein concentration of 1-20 milligrams per milliliter.
5. The cell traction measurement method according to claim 1, wherein the acquiring a displacement field comprises:
acquiring images of the substrate at different moments;
calculating the instantaneous displacement fields of the substrate at different moments;
summing the instantaneous displacement fields at different moments to obtain an accumulated deformation field; and
and acquiring a displacement field applied to the substrate by the cell to be detected according to the accumulated deformation field.
6. The method of claim 5, wherein calculating the instantaneous displacement field of the substrate at different moments in time comprises:
obtaining the similarity of the images of the substrate at different moments through a cross-correlation function; and
and acquiring the instantaneous displacement fields of the substrate at different moments according to the similarity.
7. The method of claim 1, wherein the acquiring the displacement field applied by the test cell to the substrate comprises:
step 1, taking the base image at the initial moment as a reference image, and taking the base image at the later moment as a deformed image;
step 2, dividing the reference image into a plurality of sub-images F1 with the same size as a query window, and searching a deformed sub-image F2 which is most similar to the reference sub-image F1 in the deformed image;
step 3, determining the similarity between the reference sub-image F1 and the deformed sub-image F2 through a cross-correlation function, and acquiring an instantaneous displacement field between the reference sub-image F1 and the deformed sub-image F2 according to the similarity; wherein the cross-correlation function is:
wherein F is 1 (x, y) is the image gray value of the sub-image F1 at the (x, y) coordinates; f (F) 2 (x+u dx ,y+u dy ) To have (u) with the sub-image F1 dx ,u dy ) The distorted sub-image F2 of the transient displacement field has gray values at the (x, y) coordinates; and
and 4, executing steps 2 and 3 on all the reference sub-images F1 through a grading algorithm, and obtaining a displacement field applied to the substrate by the cells to be detected.
8. The cell traction measurement method according to claim 7, wherein the grading algorithm is used to increase the calculation speed and the spatial resolution of the displacement field, comprising:
the first sub-image F1 has a size of 64×64 pixels, the second sub-image F1 has a size of 32×32 pixels, and the third sub-image F1 has a size of 16×16 pixels.
9. The cell traction measurement method of claim 1, wherein the acquiring a stress field comprises:
based on the displacement field, by two-dimensional fast Fourier transform FT 2 Function calculation of frequency domain spatial displacement fieldWherein,u (r) is the displacement field;
based on the frequency domain spatial displacement fieldBinding green's function->Calculating the spatial stress field of the frequency domain->Wherein (1)>And
based on the frequency domain spatial stress fieldBy two-dimensional inverse fast Fourier transform->Calculating a stress field by a function, wherein ∈>T (r) is the stress field applied by the test cell to the substrate.
10. The cell traction measurement method according to claim 1, wherein the obtaining a value of strain energy comprises: calculating the value of strain energy applied to the substrate by the cell to be tested through a strain energy algorithm, wherein the formula of the strain energy algorithm is as follows:
wherein,and->Respectively a stress vector and a displacement vector, A p The pixel area, U, is the value of strain energy.
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