A biological tissue section stiffness measurement method based on a photonic crystal colorimetric sensor

Abstract

The application discloses a kind of biological tissue section rigidity measurement methods based on photonic crystal colorimetric sensor, the method is according to the set step pitch vertically to photonic crystal colorimetric sensor and is pressed down to tissue section, photonic crystal colorimetric sensor is formed by the elastomer of better biocompatibility and is covered photonic crystal, its Young's modulus can be adjusted according to the rigidity range of tissue section, in the process of pressing down, the different deformation of elastomer due to the rigidity of different regions of tissue, the wavelength of internal photonic crystal reflection changes, that is, the color difference of sensor surface is generated, then the rigidity information of tissue section is obtained according to the color difference analysis.The method realizes the rigidity of biological tissue section fast, simple, high-precision detection, and can be applied to the fields such as tissue engineering, organ chip, clinical examination and the like.

Description

Technical Field

[0001] This invention relates to the field of biomechanical measurement, specifically to a method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor. Background Technology

[0002] Existing methods for measuring the stiffness of cells or tissues commonly include atomic mechanical microscopy (AFM), optical tweezers microscopy (OTM), and magnetic tweezers, each with different functions and characteristics.

[0003] Analytic perforation (AFM) is the most popular method through probe indentation, offering advantages such as highly controllable applied force, minimal sample damage, high sensitivity and spatial resolution, and ease of sample preparation. In AFM measurement of cell stiffness, a flexible silicon or silicon nitride cantilever driven by a piezoelectric actuator is vertically pressed against the cell surface. When the tip contacts the cell, the cantilever deflects, and the indentation depth and deflection are recorded using a photodetector. Finally, the cell stiffness information is obtained by fitting a force-indentation curve. Although AFM can accurately measure the stiffness of cells or tissues, the sharpness of the cantilever tip (required for high-resolution imaging) and the applied force (typically in the nN range) can cause tissue damage during mechanical interaction with soft tissues, leading to biased results. Furthermore, most AFM models used to fit the force-indentation curve assume the material is homogeneous and has a linear elastic modulus, which is not well-suited for the nonlinear elastic characteristics of cells or tissues.

[0004] Compared to AFM, OTM reduces the indentation force and detection rate, thus lowering the likelihood of biofilm rupture. OTM relies on microbead probes trapped by a highly focused laser beam. Because the probe fixation is non-mechanical and the trapping force generated by the laser beam is typically very small and easily fine-tuned, OTM detection is "gentler" than AFM. OTM uses trapped microbead probes to achieve indentation, which can be achieved by vertically moving the trapping objective or by using a piezoelectric actuator for vertical control along the Z-axis, ensuring the laser focus remains fixed as the biological tissue approaches the microbead probe. Once the biological tissue contacts the microbead, the microbead moves vertically, experiencing a force proportional to its displacement. The probe displacement is measured by a photodetector recording the deflection of the laser beam scattered by the microbead. Similar to AFM, this signal can be converted into a displacement / force curve. Although OTM can provide high-precision measurements at low forces, the technique is time-consuming due to limitations in the oscillation frequency of the microbead probe, and the heat generated by the highly focused laser beam may also have some impact on the biological tissue. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for rapidly and accurately measuring the stiffness distribution of biological tissue sections.

[0006] Technical solution: The biological tissue section stiffness measurement method based on the photonic crystal colorimetric sensor, comprising the following steps:

[0007] (1) Adjust the biological tissue section to be measured and the photonic crystal colorimetric sensor to be parallel, the photonic crystal colorimetric sensor comprising an elastomer and a photonic crystal, the elastomer covering the photonic crystal;

[0008] (2) Vertically press the biological tissue section to be measured to the surface of the photonic crystal colorimetric sensor;

[0009] (3) Vertically press the biological tissue section to be measured into the photonic crystal colorimetric sensor at a set step interval;

[0010] (4) Record the color distribution of the photonic crystal colorimetric sensor at this depth of depression using an imaging device, and observe the color change;

[0011] (5) Repeat steps (3) and (4) until the color of the photonic crystal colorimetric sensor is stable;

[0012] (6) Calculate the stiffness of the tissue section according to the elastic modulus and color distribution of the photonic crystal colorimetric sensor.

[0013] Wherein, step (6) comprises the following steps:

[0014] (61) Obtain the wavelength value λ max according to the color distribution;

[0015] (62) Obtain the change amount d pc of the adjacent particle spacing D according to the change amount of the wavelength value λ max and the formula , wherein m is the diffraction order, n avg is the average refractive index of the photonic crystal, θ is the incident angle, and d pc also represents the depth of depression at different positions of the photonic crystal colorimetric sensor;

[0016] (63) Calculate the biological tissue compression stress σ samp according to the change amount d pc of D and the formula σ samp =-(d pc ·E pc ) / H pc , wherein E pc is the elastic modulus of the elastomer, and H pc is the initial thickness of the photonic crystal colorimetric sensor;

[0017] (64) Calculate the biological tissue compression strain ε samp according to the change amount d pc of D and the formula ε samp =(d-dpc ) / H samp The biological tissue strain ε samp is calculated, where d is the depth of depression, H samp is the thickness of the biological tissue;

[0018] (65) According to the biological tissue compression stress σ samp and the biological tissue strain ε samp , a stress-strain curve characterizing the stiffness of the biological tissue slice is obtained.

[0019] Wherein, when the biological tissue slice is subjected to a smaller force or strain, its elastic behavior is close to linear elasticity, and the secant modulus E samp of the biological tissue strain ε samp = -d pc ·H samp ·E pc / (H pc ·(d-d pc )).

[0020] During the depression process, the elastomer of the photonic crystal colorimetric sensor deforms differently due to the stiffness of different regions of the tissue, the reflection wavelength of the internal photonic crystal changes, that is, the color difference on the surface of the sensor occurs. When the photonic crystal colorimetric sensor is vertically compressed, the particles inside the sensor are displaced, the distance between adjacent particles is reduced, and the reflection peak is reduced, that is, the color of the surface of the photonic crystal colorimetric sensor is blue-shifted. Then, the stiffness information of the tissue slice is obtained according to the color difference. Since the color information is intuitive, the measurement personnel can also obtain rough stiffness information by direct observation.

[0021] Wherein, in step (2), the biological tissue slice and the photonic crystal colorimetric sensor always remain parallel. The biological tissue slice remains vertically descending during the depression process.

[0022] Wherein, in step (2), the biological tissue slice and the photonic crystal colorimetric sensor are immersed in a buffer solution before depression, so that the humidity of the biological tissue is maintained during the measurement process.

[0023] Wherein, in step (2), the step distance range is 5nm-100μm. The step distance should be set reasonably so that the strain of the photonic crystal colorimetric sensor is in a reasonable range. During the depression process, the depression depth is an integer multiple of the step distance.

[0024] Wherein, the reflection wavelength of the photonic crystal is a visible light wavelength, and the wavelength range is 390nm-780nm.

[0025] The photonic crystal colorimetric sensor has a thickness greater than the pressing depth of the biological tissue section and the thickness of the biological tissue section, and the thickness of the photonic crystal colorimetric sensor is 1 μm-500 μm.

[0026] The material of the elastic body is a biocompatible material.

[0027] The present application can select the elastic body with corresponding Young's modulus according to the different stiffness ranges of the biological tissue sample.

[0028] The Young's modulus of the elastic body is 500 Pa-1 MP.

[0029] The material of the elastic body is any one of hydrogel, polyurethane, polylactic acid, polydimethylsiloxane and rubber, and the above-mentioned materials all have good biocompatibility, but the present application is not limited to the above-mentioned materials.

[0030] Advantages: compared with the prior art, the present application has the following advantages:

[0031] (1) Simplify the measurement method, directly reflect the stiffness difference of the biological tissue in the first time, and also obtain the specific stiffness information of the biological tissue according to the image and simple algorithm analysis;

[0032] (2) Without the probe, the problem of incompatibility between the sharpness of the pressing probe and the high-resolution detection in the traditional stiffness detection method is effectively solved;

[0033] (3) By adjusting the Young's modulus and size of the elastic body of the photonic crystal colorimetric sensor, the biological tissue section with different stiffness and size can be adapted, and the detection range of the biological tissue stiffness is improved;

[0034] (4) The required analysis algorithm is simple, low-noise, high-sensitivity, and the reflected light energy of the mechanical information is far lower than the laser beam of the OTM, so that the influence of the phototoxicity on the biological tissue sample is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the flow chart of the measurement method of the present application;

[0036] Fig. 2(a) is a state diagram of the biological tissue sample in the embodiment before being pressed to the photonic crystal colorimetric sensor;

[0037] Fig. 2(b) is a state diagram of the biological tissue sample pressed to the photonic crystal colorimetric sensor in the embodiment;

[0038] Fig. 3(a) is a bright field photo of the skin tissue section under the 10X objective lens of the microscope in the embodiment;

[0039] Fig. 3(b) is a fluorescence photo of the skin tissue section under the 10X objective lens of the microscope in the embodiment;

[0040] Figure 4(a) is a reflection image of the photonic crystal colorimetric sensor when the skin tissue slice just contacts the photonic crystal colorimetric sensor in the embodiment;

[0041] Figure 4(b) is a reflection image of the photonic crystal colorimetric sensor when the skin tissue slice is pressed to a depth of 5 μm in the embodiment;

[0042] Figure 4(c) is a reflection image of the photonic crystal colorimetric sensor when the skin tissue slice is pressed to a depth of 20 μm in the embodiment;

[0043] Figure 4(d) is a reflection image of the photonic crystal colorimetric sensor when the skin tissue slice is pressed to a depth of 35 μm in the embodiment;

[0044] Figure 5 Figure 5 is a thermal map of the modulus of elasticity data obtained in the embodiment. DETAILED DESCRIPTION

[0045] The technical solutions of the present application will be further described below in combination with the drawings and specific embodiments.

[0046] As shown in Figure 2(a), in the embodiment, the photonic crystal colorimetric sensor 2 is placed in the sample cell 1, and the biological tissue sample 3 is arranged directly above the photonic crystal colorimetric sensor 2, and the imaging device 4 receives the optical color information of the photonic crystal colorimetric sensor 2. In Figures 2(a) and 2(b), the direction of the arrow shows the direction of the pressure of the biological tissue sample 3, and Figure 2(a) shows that the biological tissue sample 3 has not been pressed, while in Figure 2(b), the biological tissue sample 3 has been pressed into the photonic crystal colorimetric sensor 2.

[0047] The colorimetric sensor is a sensor for analyzing substances, and the main principle is to analyze the differences of the sample by measuring the colorimetric differences between the sample and the standard substance, by using the absorption or reflection characteristics of the substance to specific wavelengths of light. The photonic crystal colorimetric sensor of the embodiment is a sensor based on photonic crystals, which can realize high-sensitivity substance detection and analysis by the absorption or reflection of the substance to specific wavelengths of light. Compared with traditional colorimetric sensors, the photonic crystal colorimetric sensor has higher sensitivity and selectivity, because the photonic crystal has a highly structured periodicity, fast response, low cost, and easy preparation.

[0048] The photonic crystal colorimetric sensor in the embodiment is composed of an elastomer material with a known initial mechanical state, such as 500 Pa to 1 MP of the brain tissue to the cartilage tissue, and a biocompatible material with a relatively larger modulus of elasticity can be selected as the sensor according to the stiffness of the tissue to be measured, so as to more accurately measure the stiffness of the biological tissue.

[0049] The thickness of the photonic crystal colorimetric sensor in this embodiment is determined according to the thickness of the tissue slice to be measured and the diameter of the cells in the tissue, and the thickness ranges from 1 μm to 500 μm.

[0050] For example, the photonic crystal colorimetric sensor 2 is composed of an acrylamide hydrogel embedded photonic crystal, wherein the Young's modulus of the acrylamide hydrogel film is 3 kPa, the initial reflection wavelength is 390 nm, and the thickness of the photonic crystal colorimetric sensor and the tissue slice is 50 μm.

[0051] In this embodiment, the biological tissue slice is pressed against the photonic crystal colorimetric sensor at a set step interval, so that the photonic crystal colorimetric sensor changes color until the color is stable, and the color distribution is recorded, and then the stiffness distribution of the biological tissue slice is calculated according to the elastic modulus and the color distribution of the photonic crystal colorimetric sensor.

[0052] Figure 1 The photonic crystal colorimetric sensor-based stiffness measurement method shown mainly includes three main processes of loading and adjusting the tissue slice, pressing the tissue slice into the photonic crystal colorimetric sensor, and recording the color change of the photonic crystal colorimetric sensor, and the operation steps are as follows:

[0053] 1. Adjust the measured skin tissue slice and the photonic crystal colorimetric sensor to be parallel

[0054] 2. Slowly press the measured skin tissue slice vertically onto the surface of the photonic crystal colorimetric sensor, and use a buffer to immerse the measured tissue slice and the photonic crystal colorimetric sensor at the same time;

[0055] 3. Press the measured tissue slice vertically into the photonic crystal colorimetric sensor according to a set step interval;

[0056] 4. Record the color change of the photonic crystal colorimetric sensor at different compression depths using a photonic crystal bioforce microscope imaging device;

[0057] 5. Repeat steps 3 and 4 until the color of the photonic crystal colorimetric sensor does not change significantly;

[0058] 6. Calculate the stiffness distribution of the tissue slice according to the known elastic modulus of the photonic crystal colorimetric sensor and the color distribution recorded at different compression depths in step 4.

[0059] In this embodiment, when the tissue slice is pressed to contact the surface of the photonic crystal colorimetric sensor, there is no obvious color change on the photonic crystal colorimetric sensor, and the tissue slice can be clearly observed under bright field. At this time, a stable step distance is set by the piezoelectric displacement table, and a cooperative control software is used. One step is pressed, and the image capture terminal records the change of the photonic crystal colorimetric sensor once, until the color change of the photonic crystal colorimetric sensor tends to be stable. At this time, the stiffness of the biological tissue is calculated according to the elastic modulus of the photonic crystal colorimetric sensor and the color distribution received by the microscope imaging device.

[0060] Specifically, the skin tissue slice to be measured is loaded on the photonic crystal colorimetric sensor. FIGS. 3(a) and 3(b) respectively show the bright field image and the fluorescence image of the skin tissue slice under the microscope 10X objective lens. When the skin tissue slice to be measured is slowly pressed onto the photonic crystal colorimetric sensor with a film thickness of 50 μm, the deformation of the photonic crystal colorimetric sensor can cause the reflection spectrum of the photonic crystal to change, that is, the color change of the photonic crystal colorimetric sensor can be observed under reflected light, and the imaging can be recorded by the image capture terminal of the microscope. FIGS. 4(a)-(d) show the color change of the photonic crystal colorimetric sensor recorded when the tissue slice just contacts the photonic crystal colorimetric sensor and is pressed by 5 μm, 20 μm and 35 μm respectively. It can be observed that the regions with different stiffness of the tissue slice present different colors on the photonic crystal colorimetric sensor.

[0061] In this embodiment, the photonic crystal colorimetric sensor is a three-dimensional structure formed by self-assembly of colloidal particles, arranged in a face-centered cubic lattice, and the close-packed face {111} can be used as a periodic ordered structure of the diffraction model, which meets the Bragg diffraction theorem modified by Snell's law, m is the diffraction order, λ is the characteristic reflection peak, the distance between adjacent particles is D, n avg is the average refractive index of the photonic crystal material, and θ is the incident angle.

[0062]

[0063] When the incident light is vertically incident, the incident angle θ = 0°. At this time, when the diffraction order m = 1, the characteristic reflection peak takes the maximum value λ max . This wavelength corresponds to the color presented by the photonic crystal colorimetric sensor under white light source illumination. When the photonic crystal colorimetric sensor is vertically compressed, the particles inside the sensor are displaced, the distance between adjacent particles D is reduced, and the reflection peak λ max is reduced, that is, the color of the surface of the photonic crystal colorimetric sensor is blue-shifted. Therefore, the sinking depth d pc of the photonic crystal colorimetric sensor at different positions can be calculated by the color change (reflection wavelength characteristic peak shift) of the surface of the photonic crystal colorimetric sensor.

[0064] Meanwhile, we use the elastomer with known elastic modulus E pc as the substrate of the photonic crystal colorimetric sensor, the Young's modulus E is defined as the ratio of the normal stress σ to the normal strain ε, if the initial thickness of the photonic crystal colorimetric sensor is H pc , then the normal strain ε pc of the photonic crystal colorimetric sensor at this point is d pc / H pc , the stress σ pc suffered by the elastomer at this point can be calculated as ε pc ·E pc =(d pc ·E pc ) / H pc .

[0065] Since the depth of the piezoelectric displacement table is known, the depth of the piezoelectric displacement table minus the depth of the photonic crystal colorimetric sensor is the compression displacement d pc of the biological tissue, d samp =(d-d pc ), the corresponding compression stress is equal to the force suffered by the photonic crystal colorimetric sensor at the corresponding position, and the direction is opposite, f samp =-f pc , so σ samp =f samp / A=-f pc / A=-σ pc =-(d pc ·E pc ) / H pc , A is the unit area.

[0066] The thickness of the biological tissue is H samp , which is set when the slice is prepared, so the strain ε samp of the biological tissue at this time is (d-d pc ) / H samp .

[0067] By continuous pressing, the displacement and stress-strain relationship of the biological tissue under different loads can be obtained, and the stress-strain curve under different loads can be drawn. It should be noted that during this process, the appropriate pressing step distance should be set, so that the strain ε pc of the photonic crystal colorimetric sensor under multiple pressing is within a reasonable range, if the strain is small, it may produce a lot of noise; if the strain is too large, it exceeds the distance D between the colloidal particles in the photonic crystal, which does not meet the Bragg's law of photonic crystal, and the strain information cannot be converted into the wavelength information of reflected light.

[0068] The stress-strain curve reflects the stiffness of the biological tissue and can also be used to calculate the elastic modulus of the biological tissue under different loads, thereby obtaining the overall elastic modulus curve of the biological tissue. Typically, the initial elastic properties of biological tissue are relatively linear, but nonlinear behavior may occur under certain loads. By applying multiple different compression depths, the behavior of the biological tissue under different loads can be observed, thus determining its nonlinear elastic properties. In this embodiment, when the biological tissue is subjected to small forces or strains, its elastic behavior is close to linear elasticity; therefore, we approximate its stiffness as the elastic modulus for simplification.

[0069] Furthermore, based on the definition of elastic modulus, the secant elastic modulus E at this strain level can be calculated. samp =σ samp / ε samp =-d pc ·H samp ·E pc / (H pc ·(dd pc For example, in this embodiment, when the compression depth is 75 μm and the tissue is compressed (i.e., the strain is around 20 μm), the elastic modulus of the biological tissue, plotted based on calculated data, is shown in the following heat map. Figure 5 As shown.

Claims

1. A method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor, characterized in that, Includes the following steps: (1) Adjust the biological tissue slice to be tested and the photonic crystal colorimetric sensor to be in a parallel state. The photonic crystal colorimetric sensor includes an elastomer and a photonic crystal, with the elastomer covering the photonic crystal. (2) Press the biological tissue slice to be tested vertically down onto the surface of the photonic crystal colorimetric sensor; (3) Press the biological tissue slice to be tested vertically into the photonic crystal colorimetric sensor according to the set step interval; (4) Use an imaging device to record the color distribution of the photonic crystal colorimetric sensor at this pressure depth and observe its color changes; (5) Repeat steps (3) and (4) until the color of the photonic crystal colorimetric sensor is stable; (6) The stiffness of the tissue section is calculated based on the elastic modulus and color distribution of the photonic crystal colorimetric sensor; specifically, the following steps are included: (61) Obtain the wavelength value λ based on the color distribution. max ; (62) Based on the wavelength value λ max Changes and formulas Obtain the change d of the distance D between adjacent particles pc Where m is the diffraction order, n avg θ is the average refractive index of the photonic crystal, and θ is the incident angle. (63) Based on the change in D, d pc and formula σ samp =-(d pc ·E pc ) / H pc The compressive stress σ of biological tissue was calculated. samp E pc H is the elastic modulus of the elastomer. pc The initial thickness of the photonic crystal colorimetric sensor; (64) Based on the change in D, d pc and the formula ε samp =(dd pc ) / H samp The strain ε of biological tissue was calculated. samp Where d is the compression depth, and H samp Thickness of biological tissue; (65) Based on the compressive stress σ of biological tissue samp and biological tissue strain ε samp The stress-strain curves characterizing the stiffness of the biological tissue slices were obtained.

2. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that: When subjected to relatively small forces or strains, the elastic behavior of the biological tissue slices is close to linear elasticity, and the strain ε of the biological tissue is... samp Secant modulus E samp =-d pc ·H samp ·E pc / (H pc ·(dd pc )).

3. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that, In step (2), the biological tissue slice and the photonic crystal colorimetric sensor are kept parallel at all times.

4. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that, In step (2), the biological tissue slices to be tested and the photonic crystal colorimetric sensor are immersed in buffer solution before pressing down.

5. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that, In step (2), the step spacing ranges from 5nm to 100μm.

6. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that, The photonic crystal reflects light at the visible wavelength.

7. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that: The thickness of the photonic crystal colorimetric sensor is greater than the indentation depth of the biological tissue slice and the thickness of the biological tissue slice, and the thickness of the photonic crystal colorimetric sensor is 1μm to 500μm.

8. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 1, characterized in that, The elastomer is made of a biocompatible material, and the Young's modulus of the elastomer is 500 Pa to 1 MPa.

9. The method for measuring the stiffness of biological tissue sections based on a photonic crystal colorimetric sensor according to claim 8, characterized in that, The elastomer is made of any one of hydrogel, polyurethane, polylactic acid, polydimethylsiloxane, and rubber.

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