WO2020155324A1 - 多模态生物力学显微镜及测量方法 - Google Patents

多模态生物力学显微镜及测量方法 Download PDF

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WO2020155324A1
WO2020155324A1 PCT/CN2019/078319 CN2019078319W WO2020155324A1 WO 2020155324 A1 WO2020155324 A1 WO 2020155324A1 CN 2019078319 W CN2019078319 W CN 2019078319W WO 2020155324 A1 WO2020155324 A1 WO 2020155324A1
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photonic crystal
hydrogel film
crystal hydrogel
cell
film
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French (fr)
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顾忠泽
李奇维
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东南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection

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  • the invention relates to a cell traction force microscope and a measurement method, in particular to a multimodal biomechanical microscope and a measurement method.
  • CTF cell traction
  • CTF Cells also use CTF to sense the mechanical properties of the underlying substrate and adjust their adhesion and morphology.
  • CTF is used to control cell shape and maintain the steady state of cell tension. Therefore, CTF is necessary for many basic biological processes, including morphogenesis, metastasis, angiogenesis, and wound healing.
  • CTF is also necessary for mechanical signal transmission and transduction. Since CTF is transmitted to ECM through FAs, FA is composed of a variety of proteins, including signal proteins (such as integrins) and enzymes (such as kinases and phosphatases).
  • CTF can deform the ECM network, thereby generating stress and strain in the matrix network, which in turn can regulate cell functions, such as DNA synthesis, ECM protein secretion, and even cell differentiation. Therefore, CTF can also be used as a useful "biophysical marker" for characterizing the phenotypic changes of individual cells. In short, careful study of CTF can better understand the cellular and molecular mechanisms of many important biological processes.
  • CTF measurement there are many methods for CTF measurement, such as cell traction force microscope, micro-pillar matrix and fluorescence resonance energy transfer, which have different functions and characteristics.
  • the cell traction force microscopy technology is to record the microscopic image of the displacement of fluorescent particles caused by the elastic deformation of the substrate caused by the elastic deformation of the substrate during the shrinkage or migration of the cells cultured on the polyacrylamide gel elastic substrate mixed with randomly distributed fluorescent microparticles. Then use the digital image correlation method based on the fast recursion relationship to track the changes of the fluorescent particle pattern to obtain the elastic deformation field of the substrate, and then invert the corresponding cell traction force field.
  • the cell traction force microscope is the first and most widely used single-cell traction force measurement method, however, due to the pathological characteristics of the inverse problem, it is usually extremely difficult and computationally expensive to obtain an accurate cell traction force field from the basal displacement field.
  • the previous methods mainly used complex and cumbersome regularization processing to improve the reliability and accuracy of force inversion.
  • the key to regularization processing lies in the selection of appropriate regularization parameters. This requires the use of as much prior knowledge as possible in combination with specific algorithms to achieve. Generally speaking, the selection of regularization parameters is more or less subjective. For color, the process of selecting the optimal value of the regularization parameter is relatively difficult. This disadvantage of regularization processing makes the traction inversion efficiency of the previous calculation methods extremely low, which is not conducive to the statistical analysis of a large number of data in the cell experiment, and the inversion accuracy is also low, even if the inversion of the optimal regularization parameter value is obtained The results are sometimes far from the real situation.
  • the microbeads are just at the position where the traction force is balanced, such as the midpoint of two cells that apply the same force to the ECM, since the microbeads are not displaced, the wrong result will be that there is no traction force at this point. Also, because the resolution of the commonly used microscopic imaging system in the Z direction is much lower than that in the XY direction, the final stress field lacks the resolution in the Z direction.
  • Micropillar array sensors are a simpler option.
  • the micro-pillar array is usually made of flexible polymer materials with carefully designed micron-level column heights and column spacing. Each column is like a mini spring. By measuring its deflection, the force exerted by the cell can be identified and measured.
  • Micropillar array data are easier to interpret than the results of TFM experiments, and they require less computational analysis.
  • the device itself is quite simple to manufacture and compatible with fluorescence microscopes. However, these arrays impose a specific and unnatural pattern between the cells and the substrate, which is likely to make the behavior of the cells different from those in the organism.
  • Fluorescence resonance energy transfer technology has also been applied to the measurement of cell traction in recent years.
  • the optical resolution is sufficiently high , In theory, sub-micron level mechanical measurement can be achieved.
  • other protein molecules can also be selected to obtain different sensitivity.
  • the design and verification of this sensor requires a lot of energy, and the sensor may "dark" due to reasons other than force detection, such as degradation.
  • FRET signals are also difficult to interpret and require careful measurement to eliminate false alarms. Inserting fluorescent molecules into a protein whose function is strongly dependent on structure may also affect its function. Generally speaking, it is difficult to predict how much this effect will be.
  • the objective of the present invention is to provide a multi-modal biomechanical microscope and measurement method that can measure cell traction in real time, solve the problem of rapid and high-throughput measurement of cell traction, and can realize traditional image information and mechanical information Multi-modal imaging.
  • the multi-modal biomechanical microscope of the present invention includes a transmission light source, a photonic crystal hydrogel film, a stage, a reflection light source, and an imaging component that are sequentially arranged from top to bottom;
  • the transmission light source is used for waiting Measuring cell morphology imaging;
  • the photonic crystal hydrogel film is used as a substrate for cell culture to be tested and a sensor for measuring cell traction;
  • the reflected light source is used to measure the mechanical information of the cell to be tested, and the spectrum of the reflected light source contains photonic crystal water Bandwidth range of reflection peak of gel film;
  • the photonic crystal hydrogel film is placed on the stage and kept in a suspended state, so that the transmitted light can penetrate the film to reach the imaging component, so that the reflected light reaches the imaging component after being reflected by the film; when the cell to be measured is placed in the photon After the crystalline hydrogel film is attached, the photonic crystal hydrogel film supporting the cell to be tested deforms, so that the direction of the reflected light on the photonic crystal hydrogel film is changed, and the imaging component collects the reflected light and the transmitted light and Perform imaging to obtain shadow images that characterize cell traction.
  • the strongest reflection waveband reflectivity of the photonic crystal hydrogel film in the 450-800nm waveband is greater than 35%, and the light transmittance in the non-gap range is greater than 80%. To obtain a higher signal-to-noise ratio.
  • the thickness of the photonic crystal hydrogel film is 5-200 microns.
  • the Young's modulus of the photonic crystal hydrogel material is 1-100 kPa.
  • the strongest reflection wavelength of the photonic crystal hydrogel material is 450-800 nanometers.
  • the microscope further includes an objective lens, the numerical aperture of the objective lens is 0.1-0.9, and the magnification is 2-60 times.
  • the present invention also provides a multi-modal biomechanics measurement method, including the following steps:
  • step (2) Place the sample obtained in step (1) on the stage of the microscope, and the photonic crystal hydrogel film deforms under the action of the cell to be tested to change the direction of the incident reflected light, thereby obtaining a shadow characterizing the traction of the cell image;
  • the traction force distribution of the cell to be tested is obtained through an algorithm.
  • the multi-modal biomechanical microscope provided by the present invention can obtain information on mechanical modes, morphological modes, such as phase difference, DIC, and bright field. After the cells to be tested are placed on the photonic crystal material of the multimodal biomechanical microscope of the present invention for culture, the photonic crystal film as the growth substrate for the cells to be tested is bent by the cell pulling force exerted by the cells, which changes the surface of the photonic crystal film.
  • the normal vector direction deflects the direction of the scattered part of the light projected by the reflected light source on the photonic crystal material, so that the image obtained in the microscope camera becomes darker in these areas, and conforms to the law that the more the deflection, the darker the image, namely
  • the brightness of a specific pixel of an image is a function of the angle between the normal vector of the corresponding point of the photonic crystal and the main optical axis. Therefore, the local cell traction can be judged intuitively from the brightness of the image.
  • the morphological change of the photonic crystal film can be obtained through the algorithm of restoring the morphology from the shadow in computer vision, thereby obtaining the distribution of cell traction more accurately.
  • the cell traction force measurement is based on the shadow image formed by the photonic crystal material reflecting the objective lens light source, and the deformation of the photonic crystal material caused by the cell traction force is obtained in real time, with high accuracy.
  • the multi-modal biomechanics microscope and measurement method provided by the present invention can not only measure cell traction force, but also measure other biomechanics of cells based on the above-mentioned principle.
  • the first mode Repeated imaging of the area of interest before the cells adhere to the wall until the end of the experiment to determine the movement trajectory of the beads that reflect local stress changes. If you move away and then return to the field of view, it may not be possible to infer the corresponding relationship between the beads in the current field of view and the beads in the original state and the stress distribution cannot be obtained; the second mode: fluorescence imaging of the sample of interest to determine the beads at the current moment After position, trypsin is added to separate the cells and the substrate.
  • the imaging is continued to obtain the movement trajectory of the beads during the process from the uneven stress distribution state to the unstressed state of the substrate to obtain the corresponding relationship of the beads between the two states.
  • the first mode it is difficult to use the same optical system to image a large number of samples; in the second mode, because trypsin digestion will damage the sample, it is impossible to perform multiple measurements on the same sample of interest.
  • the multi-modal biomechanical microscope provided by the present invention does not have such a limitation, because the initial state of the photonic crystal hydrogel film as the sensing unit is known, and there is no need to construct a corresponding relationship.
  • the cell traction force microscope faces high-speed changing samples, such as beating myocardium. Due to the weak fluorescence of fluorescent beads, in order to obtain sufficiently high time resolution, high-energy excitation light and highly sensitive microscope cameras are required, which may cause damage to the samples. At the same time of damage, there are higher requirements for the experimental hardware.
  • the mechanical information in the multi-modal biomechanics microscope provided by the present invention is contained in the reflected light, and its brightness is usually much higher than that of fluorescence. Therefore, there is no need to increase the light intensity of the light source and use a highly sensitive microscope camera to better maintain The original state of the sample is improved, and the requirements for the microscope camera are greatly reduced.
  • the present invention proposes a method for spatial quantitative measurement of cell biomechanics using a photonic crystal film.
  • the photonic crystal film is imaged to obtain shadow or color information, and the local normal vector information of the surface is obtained according to the model and algorithm, and then reconstructed The entire surface; real-time measurement of cell traction, realizing multi-modal imaging of traditional image information and mechanical information.
  • the mechanical information is contained in the reflected light, and its brightness is usually much higher than that of fluorescence, so there is no need to increase the light intensity of the light source and use a highly sensitive microscope camera.
  • the ground maintains the original state of the sample while greatly reducing the requirements for the camera in the microscope.
  • Figure 1 is a schematic diagram of the structure of a multimodal biomechanics microscope
  • Figure 2 is a schematic diagram of the reflected light effect of the photonic crystal hydrogel film under the action of the cell traction force
  • Figure 3 is a schematic diagram of the reflected light action of the photonic crystal hydrogel film under the action of cell traction
  • Figure 4-1 is the mechanical modal imaging picture when pancreatin is added for 30s;
  • Figure 4-2 is the co-imaging picture of mechanical mode and phase difference mode when pancreatin is added for 30s;
  • Figure 4-3 is the surface topography of the film reconstructed from the mechanical modal information when pancreatin is added for 30s;
  • Figure 5-1 is the mechanical modal imaging picture when pancreatin is added for 60s;
  • Figure 5-2 is the co-imaging picture of the mechanical mode and the phase difference mode when pancreatin is added for 60s;
  • Figure 5-3 is the surface topography of the film reconstructed from the mechanical modal information when pancreatin is added for 60s;
  • Figure 6-1 is the mechanical modal imaging picture when pancreatin is added for 90s
  • Figure 6-2 is the co-imaging picture of the mechanical mode and the phase difference mode when pancreatin is added for 90s;
  • Figure 6-3 is the surface topography of the film reconstructed from the mechanical modal information when pancreatin was added for 90s.
  • the multi-modal biomechanical microscope of the present invention includes a transmission light source 1, a photonic crystal hydrogel film 2, a stage 3, a reflective light source 4, and an imaging component arranged in order from top to bottom.
  • the imaging component includes Objective lens 6, beam splitter 8, CCD camera 9.
  • the transmission light source 1 is used for morphological imaging of the cells to be tested;
  • the photonic crystal hydrogel film 2 is used as a substrate for the cell culture to be tested and a sensor for measuring cell traction;
  • the spectrum includes the strongest reflection band of the photonic crystal hydrogel film 2.
  • the photonic crystal hydrogel film 2 is placed on the stage 3 and kept in a suspended state, so that the transmitted light can penetrate the film to reach the imaging component, and the reflected light can reach the imaging component after being reflected by the film.
  • the photonic crystal hydrogel film 2 supporting the cell to be tested is deformed, so that the direction of the reflected light on the photonic crystal hydrogel film 2 changes.
  • the component collects reflected light and transmitted light and performs imaging to obtain shadow images that characterize cell traction.
  • Figure 2 shows a schematic diagram of the reflected light effect of the photonic crystal hydrogel film 2 without the traction force of the cells. It can be seen that the light emitted from the reflected light source interacts with the photonic crystal hydrogel film 2 and is mainly divided into Transmitting and reflecting two parts, the photonic crystal hydrogel film 2 is horizontal on the support without cell traction, and the normal direction of each point on the surface is basically parallel to the main optical axis direction of the objective lens, so the microscope In the field of view, the reflected light intensity of each point of the photonic crystal film is almost the same, and there is no shadow.
  • a circular polyacrylamide photonic crystal hydrogel film 2 with a diameter of 1 cm and a thickness of 20 microns is fixed on the support 7 so that the film is in a suspended state, and the polyacrylamide photonic crystal hydrogel film 2 together with the support 7 Put them together in a container.
  • the Young's modulus of the polyacrylamide photonic crystal hydrogel film 2 is 20kPa, which is suitable for the growth of primary fibroblasts from the heart;
  • the central reflection wavelength of the photonic crystal film is at 600nm, which is convenient for using a longer wavelength light source for illumination.
  • the half-width of the reflection peak is 20nm
  • the reflectance of the strongest reflection band is 40%
  • the light transmittance in the non-gap range is greater than 85% to obtain a higher signal-to-noise ratio.
  • Fig. 3 is a schematic diagram of the reflected light action of the photonic crystal hydrogel film 2 under the action of the cell traction.
  • the photonic crystal hydrogel film 2 deforms under the action of the cell to be tested, which changes the direction of the incident reflected light, and then
  • the imaging component obtains shadow images that characterize cell traction.
  • the growth of fibroblasts on the photonic crystal film exerts a traction force on the photonic crystal film, causing the photonic crystal film to bend and form folds or protrusions.
  • the photonic crystal In addition to the wavelength selectivity of the light reflected by the photonic crystal, it also has the characteristics of a mirror surface, that is, the incident light and the reflected light are symmetrically distributed with the normal to the point. Therefore, the greater the traction force exerted by the cells on the photonic crystal hydrogel film 2 and the greater the inclination angle formed by the film with the initial parallel position, more light will be reflected to directions other than the objective lens, which is also reflected in the pattern captured by the camera. The darker.
  • the photonic crystal hydrogel film 2 is placed on the stage 3 perpendicular to the main optical axis.
  • the transmitted light and the reflected light can be transmitted and reflected on the photonic crystal hydrogel film well, so that the image quality obtained is better.
  • Configure the beam splitter to be semi-transparent and semi-reflective for visible light. Turn on the fluorescent excitation light source of the halogen tungsten lamp as the reflected light source and turn on the fluorescent shutter. Turn on the phase difference light source as the transmission light source, and adjust the brightness of the phase difference light source and the fluorescence excitation light source.
  • the median filter is used to preprocess the mechanical modal photos, and the median filter template size is 10*10. Since the law of light and dark changes is similar to the Lambert model, the image processing method mentioned by Emmanuel Prados in the DOI:10.1007/s10851-006-6899-x paper can be used to process the image to obtain the three-dimensional shape of the photonic crystal film surface.
  • the appearance information reflects the size of the force that the cells exert on the substrate. In this way, combined with the phase-difference images taken before, the multi-modal information of the cell morphology and mechanics of the area is obtained, and then the traction force distribution of the cell to be tested is obtained.
  • the primary fibroblasts of neonatal mouse myocardium were cultured on polyacrylamide photonic crystal hydrogel film and then digested with trypsin, as shown in Figure 4-1, 4-2, and 4-3 are the mechanics when trypsin was added for 30 seconds.
  • Modal imaging pictures, co-imaging pictures of mechanical modal and phase difference modal, as well as the surface topography of the film reconstructed from the mechanical modal information are respectively when pancreatin is added for 60s
  • the structure of the microscope in this embodiment is the same as that in embodiment 1.
  • the photonic crystal hydrogel film has a Young's modulus of 30kPa, a central reflection wavelength of 600nm, a reflection peak half-width of 20nm, and a strongest reflection band reflectance of 40%
  • a polyacrylamide photonic crystal hydrogel film with a thickness of 20 microns and a diameter of 1 cm with a light transmittance of greater than 85% in the non-forbidden band range is fixed on the support, and the polyacrylamide photonic crystal hydrogel film together with the support Put it in a container.
  • the cultured cell culture dish is taken out and placed on the stage 3 of the microscope, and the beam splitter is configured to be semi-transparent and semi-reflective for visible light.
  • Turn on the phase difference light source as the transmission light source and adjust the brightness of the phase difference light source and the fluorescence excitation light source.
  • Turn off the transmission light source use the microscope camera to record the area to be studied at a frame rate of 30 frames with a resolution of 800*600 to obtain mechanical modal information.
  • Turn off the fluorescent shutter that is, turn off the reflected light source.
  • Turn on the transmission light source use the microscope camera to record the area to be studied at a frame rate of 30 frames with a resolution of 800*600, and obtain general phase difference modal information.
  • the median filter is used to preprocess the mechanical modal photos, and the median filter template size is 10*10.
  • the image is processed to obtain the three-dimensional topography information of the photonic crystal film surface, which reflects the force of the cell on the substrate. Since the intensity of the reflected light is determined by the reflectance of the photonic crystal hydrogel film and the light intensity of the reflected light source, and because the photonic crystal hydrogel we use has a reflectivity of up to 60% at the strongest reflection peak, it can be used Traditional CMOS or CCD camera continuous mechanical modal information.

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Abstract

一种多模态生物力学显微镜及测量方法，包括从上至下依次设置的透射光源(1)、光子晶体水凝胶薄膜(2)、载物台(3)、反射光源(4)、成像组件；光子晶体水凝胶薄膜(2)置于载物台(3)上并保持悬空状态，使得透射光可以穿透光子晶体水凝胶薄膜(2)到达成像组件，反射光经光子晶体水凝胶薄膜(2)反射后到达成像组件；当待测细胞置于光子晶体水凝胶薄膜(2)上后，支撑待测细胞的光子晶体水凝胶薄膜(2)发生形变，使得光子晶体水凝胶薄膜(2)上的反射光方向发生改变，成像组件收集反射光和透射光并进行成像，得到表征细胞牵引力的阴影图像。能够对细胞牵引力进行实时测量，实现传统图像信息与力学信息的多模态成像。

Description

多模态生物力学显微镜及测量方法 技术领域
本发明涉及细胞牵引力显微镜及测量方法，特别是涉及一种多模态生物力学显微镜及测量方法。
背景技术
细胞力在许多基本的生物过程中起着至关重要的作用。各种生命活动，包括心跳和身体运动，都依赖于肌肉收缩，肌肉收缩最终由个体肌肉细胞的内在收缩决定。在肌细胞中，细胞收缩是由肌球蛋白在肌动蛋白丝上连续高速滑动产生的；在非肌肉细胞中，类似的机制被用于产生细胞内张力。当这种细胞内张力通过局部锚定点(FAs)传递到细胞外基质(ECM)时，在肌动蛋白细胞骨架和ECM之间建立物理联系，这种力被称为细胞牵引力(CTF)。CTF在细胞活动的许多方面都很重要。细胞在其底层基质上应用CTF以使细胞迁移。细胞还使用CTF来感测其下面的基底的机械性质并调节它们的粘附性和形态。此外，CTF用于控制细胞形状和维持细胞张力稳态。因此，CTF是许多基本生物学过程所必需的，包括形态发生，转移，血管生成和伤口愈合。此外，CTF也是机械信号传输和转导所必需的。由于CTF通过FAs传递给ECM，FA由多种蛋白质组成，包括信号蛋白(如整合素)和酶(如激酶和磷酸酶)，任何通过ECM作用于细胞的生物、生物化学或生物力学刺激都可能导致FA蛋白、肌动蛋白细胞骨架和肌动蛋白相互作用的变化，这些变化反过来会影响CTF的“输出”。另一方面，CTF可以使ECM网络变形，从而在基质网络中产生应激和应变，进而可以调节细胞功能，如DNA合成，ECM蛋白分泌，甚至细胞分化。因此，CTF还可以用作表征单个细胞的表型变化的有用的“生物物理标记”。总之，仔细研究CTF可以更好地理解许多重要生物过程的细胞和分子机制。
目前已有多种方法来进行CTF的测量，如细胞牵引力显微镜、微柱矩阵和荧光共振能量转移等，具有不同的功能和特点。
细胞牵引力显微镜技术是通过记录培养在掺有随机分布的荧光微颗粒的聚丙烯酰胺凝胶弹性基底上的细胞在收缩或迁移过程中引起的基底弹性变形而引起的荧光颗粒位移的显微图像，再使用基于快速递推关系的数字图像相关方法，跟踪这些荧光颗粒图案的变化，以此获得基底弹性变形场，进而反演求解相应的细胞牵引力场。尽管细胞牵引力显微镜是第一个也是最为广泛使用的单细胞牵引力测试方法，然而，由于反问题的病态特点，从基底位移场获得准确的细胞牵引力场通常极为困难且计算量巨大，很大程度上是由于荧光微珠初始状态的随机性所导致的。以往的方法主要采用复杂繁琐的正则化处理来提高力反演的可靠性和精度。正则化处理的关键在于合适正则化参数的选取，这需要运用尽可能多的先验知识并结合特定的算法来实现，一般来讲，正则化参数的选取或 多或少都带有一定的主观色彩，选取正则化参数最优值的过程相对困难。正则化处理的这一劣势使得以往计算方法的牵引力反演效率极低，不利于细胞实验中大量数据统计分析，同时反演精度也较低，即使通过最优的正则化参数值反演得到的结果有时也与真实情况差距较大。此外，如果微珠刚好位于牵引力平衡的位置，如两个个向ECM施加同样大小的力的细胞的中点，由于微珠没有位移，将得到该点没有牵引力的错误结果。还由于通常使用的显微成像系统在Z方向的分辨率远低于XY方向，使得最终的应力场缺乏Z方向的分辨率。
微柱阵列传感器是一种更简单的选择。微柱阵列通常是由柔性聚合物材料制成，其具有精心设计的微米级的柱高和柱间距。每根柱子就像一根迷你弹簧，通过测量其偏转，便可以识别并测量细胞在上面施加的力。微柱阵列数据比TFM实验的结果更容易解释，并且它们需要较少的计算分析。而且设备本身制造起来相当简单，且与荧光显微镜兼容。然而，这些阵列给细胞和基底之间强加了一种特定且非自然的模式，这很可能使得细胞的行为和在生物体内有较大的差异。
荧光共振能量转移技术在近年也被应用到了细胞牵引力的测量中。通过将荧光分子连接到一种具有优良弹性的蛛丝蛋白分子上，通过观察这两个荧光基团间的共振能量转移现象并结合蛛丝蛋白的力学信息，在光学分辨率足够高的情况下，理论上可以实现亚微米级别的力学测量。根据需要，也可以选择别的蛋白分子来获得不同的灵敏度。但是这种传感器的设计和验证需要耗费大量的精力，并且传感器可能因为力检测以外的原因而“变暗”，如降解等。FRET信号也很难解释，需要仔细测量以消除误报。将荧光分子插入到功能强烈依赖于结构的蛋白质中还可能会对其功能产生影响，而通常来说，这种影响究竟有多大是很难预测的。
发明内容
发明目的：本发明的目的是提供一种多模态生物力学显微镜及测量方法，能够对细胞牵引力进行实时测量，解决细胞牵引力的快速与高通量测量的问题，能够实现传统图像信息与力学信息的多模态成像。
技术方案：本发明所述的多模态生物力学显微镜，包括从上至下依次设置的透射光源、光子晶体水凝胶薄膜、载物台、反射光源、成像组件；所述透射光源用于待测细胞形态成像；所述光子晶体水凝胶薄膜用作待测细胞培养的基底和测量细胞牵引力的传感器；所述反射光源用于测量待测细胞力学信息，并且反射光源的光谱包含光子晶体水凝胶薄膜的反射峰带宽范围；
所述光子晶体水凝胶薄膜置于载物台上并保持悬空状态，使得透射光可以穿透薄膜到达成像组件，使得反射光经所述薄膜反射后到达成像组件；当待测细胞置于光子晶体水凝胶薄膜上后，支撑所述待测细胞的光子晶体水凝胶薄膜发生形变，使得光子晶体水 凝胶薄膜上的反射光方向发生改变，所述成像组件收集反射光和透射光并进行成像，得到表征细胞牵引力的阴影图像。
优化地，所述光子晶体水凝胶薄膜在450-800nm波段内的最强反射波段反射率大于35％，非禁带范围透光率大于80％。以获得较高的信噪比。
为了能够更好地对针对待测细胞进行细胞牵引力的测量，使待测细胞能够产生足够的形变，所述光子晶体水凝胶薄膜的厚度为5-200微米。
所述光子晶体水凝胶材料杨氏模量为1-100kPa。
所述光子晶体水凝胶材料的最强反射波长为450-800纳米。
所述显微镜还包括物镜，所述物镜的数值孔径为0.1-0.9，放大倍率为2-60倍。
本发明还提供了一种多模态生物力学测量方法，包括以下步骤：
(1)将本发明中的多模态生物力学显微镜中的光子晶体水凝胶薄膜进行前处理，后将待测细胞置于光子晶体水凝胶薄膜上进行培养；
(2)将步骤(1)得到的样品置于显微镜的载物台上，光子晶体水凝胶薄膜在待测细胞作用下发生形变而使入射的反射光方向变化，进而获得表征细胞牵引力的阴影图像；
(3)根据阴影图像通过算法得到待测细胞的牵引力分布。
发明原理：本发明提供的多模态生物力学显微镜，可进行力学模态、形态模态，如相差、DIC、明场等的信息获取。将待测细胞置于本发明中多模态生物力学显微镜的光子晶体材料上进行培养后，作为待测细胞生长基底的光子晶体薄膜受到细胞施加的细胞牵引力而弯曲，改变了光子晶体薄膜表面的法向量方向，使得反射光源投射在光子晶体材料上的光的被散射部分的方向发生偏转，使得在显微镜相机中获得的图像在这些区域变暗，且符合偏转越多图像越暗的规律，即图像特定像素的明暗是光子晶体相应点法向量和主光轴夹角的函数。从而可直观的从图像的明暗来判断局部细胞牵引力的大小。进一步，通过计算机视觉中从阴影还原形态的算法可以得到光子晶体薄膜的形态变化，从而更准确的得到细胞牵引力的分布。本发明中细胞牵引力的测量根据光子晶体材料反射物镜光源形成的阴影图像，实时获取表征细胞牵引力引起的光子晶体材料形变，准确度高。
本发明提供的多模态生物力学显微镜及测量方法不仅可以针对细胞牵引力进行测量，还可以基于上述原理对细胞的其他生物力学进行测量。
现有细胞牵引力显微镜常用使用模式有两种，目的都是为了解决测量点状态和原始无应力状态间微珠的对应关系。第一种模式：在细胞贴壁前便开始对感兴趣的区域反复成像，直到实验结束，以确定反应局部应力变化的微珠的移动轨迹。如果移开后再回到该视野，可能出现无法推断当前视野微珠和原始状态微珠的对应关系而无法得到应力分布；第二种模式：对感兴趣的样本进行荧光成像确定当前时刻微珠位置后，加入胰酶使得细胞和基底之间分离。在这个过程中持续成像，获得基底从应力分布不均匀状态到无 应力状态过程中微珠的移动轨迹从而得到两个状态间微珠的对应关系。显然，在第一种模式中，难以使用同一光学系统对大量的样本进行成像；在第二种模式中，由于胰酶消化会损坏样本，导致无法对同一感兴趣的样本进行前后多次测量。而本发明提供的多模态生物力学显微镜进行测试，没有这样的限制，因为作为传感单元的光子晶体水凝胶薄膜的初始状态是已知的，不需要进行对应关系的构建。
细胞牵引力显微镜面对高速变化的样本，如跳动的心肌，由于荧光微珠的荧光较弱，为了获得足够高的时间分辨率需要使用高能量的激发光和高灵敏的显微镜相机，对样本可能造成损伤的同时也对实验的硬件有较高的要求。而本发明提供的多模态生物力学显微镜中的力学信息是蕴含在反射光中，其亮度通常远高于荧光，因此不需要额外增加光源的光强和使用高灵敏显微镜相机，更好地维持了样本的原始状态，同时大幅降低了对显微镜相机的要求。
有益效果：(1)本发明提出了使用光子晶体薄膜对细胞的生物力学进行空间定量测量的方法，对光子晶体薄膜成像获得阴影或颜色信息，根据模型和算法获得曲面局部法向量信息进而重建出整个曲面；能够对细胞牵引力进行实时测量，实现传统图像信息与力学信息的多模态成像。(2)与目前最常用的细胞牵引力显微镜技术相比，和随机分布的荧光微珠不同，光子晶体的光学特点使得测量过程中初始状态稳定已知，大大简化了算法的复杂度、噪声的敏感度与实验的复杂度；相较于微柱阵列技术，光子晶体基底的均匀性对细胞本身的生理活动的干扰远小于离散的微柱阵列，能实现更准确的生理环境的还原以得到更准确的结果。(3)本发明中作为传感单元的光子晶体水凝胶薄膜的初始状态是已知的，不需要进行对应关系的构建。(4)本发明提供的显微镜在测量生物力学的过程中，力学信息蕴含在反射光中，其亮度通常远高于荧光，因此不需要额外增加光源的光强和使用高灵敏显微镜相机，更好地维持了样本的原始状态，同时大幅降低了对显微镜中相机的要求。
附图说明
图1是多模态生物力学显微镜的结构示意图；
图2是不受细胞牵引力作用下的光子晶体水凝胶薄膜的反射光作用示意图；
图3是受细胞牵引力作用下的光子晶体水凝胶薄膜的反射光作用示意图；
图4-1是胰酶加入30s时的力学模态成像图片；
图4-2是胰酶加入30s时的力学模态和相差模态共成像图片；
图4-3是胰酶加入30s时的力学模态信息重建出的薄膜表面形貌图；
图5-1是胰酶加入60s时的力学模态成像图片；
图5-2是胰酶加入60s时的力学模态和相差模态共成像图片；
图5-3是胰酶加入60s时的力学模态信息重建出的薄膜表面形貌图；
图6-1是胰酶加入90s时的力学模态成像图片；
图6-2是胰酶加入90s时的力学模态和相差模态共成像图片；
图6-3是胰酶加入90s时的力学模态信息重建出的薄膜表面形貌图。
具体实施方式
下面结合附图对本发明作更进一步的说明。
实施例1：
如图1所示，本发明的多模态生物力学显微镜包括从上至下依次设置的透射光源1、光子晶体水凝胶薄膜2、载物台3、反射光源4、成像组件，成像组件包括物镜6、分束镜8、CCD相机9。透射光源1用于待测细胞形态成像；光子晶体水凝胶薄膜2用作待测细胞培养的基底和测量细胞牵引力的传感器；反射光源4用于测量待测细胞力学信息，并且反射光源4的光谱包括光子晶体水凝胶薄膜2的最强反射波段。其中光子晶体水凝胶薄膜2置于载物台3上并保持悬空状态，使得透射光可以穿透薄膜到达成像组件，使得反射光经所述薄膜反射后到达成像组件。
当待测细胞置于光子晶体水凝胶薄膜2上后，支撑待测细胞的光子晶体水凝胶薄膜2发生形变，使得光子晶体水凝胶薄膜2上的反射光方向发生改变，所述成像组件收集反射光和透射光并进行成像，得到表征细胞牵引力的阴影图像。
如图2所示为不受细胞牵引力作用下的光子晶体水凝胶薄膜2的反射光作用示意图，可以看到，从反射光源发出的光和光子晶体水凝胶薄膜2作用后，主要分为透射和反射两部分，光子晶体水凝胶薄膜2在不受细胞牵引力的情况下，在支架上呈水平状态，表面每个点的法线方向基本和物镜主光轴方向平行，因而此时显微镜的视野内光子晶体薄膜每个点的反射光光强几乎一样，没有阴影。
将直径为1厘米，厚度为20微米的圆形聚丙烯酰胺光子晶体水凝胶薄膜2固定在支架7上，使得薄膜处于悬空状态，并将聚丙烯酰胺光子晶体水凝胶薄膜2连同支架7一起放在容器中。其中聚丙烯酰胺光子晶体水凝胶薄膜2的杨氏模量为20kPa，适合来自心脏的原代成纤维细胞生长；光子晶体薄膜的中心反射波长位于600nm，便于使用较长波长的光源进行光照，减小对细胞的光毒性；反射峰半峰宽为20nm，最强反射波段反射率40％，非禁带范围透光率大于85％，以获得较高的信噪比。使用Sulfo-SANPAH活化光子晶体水凝胶表面的氨基后，使用HEPES缓冲液清洗三次，随后浸泡在HEPES缓冲的0.01mg/mL的一型胶原蛋白溶液中，4摄氏度过夜。将前述光子晶体水凝胶薄膜浸泡在DMEM培养基中过夜后移到细胞培养皿中，再快速加入5％胎牛血清的DMEM培养基浸没光子晶体水凝胶。加入新生SD大鼠心脏原代成纤维细胞在5％胎牛血清的DMEM培养基中的分散液，置于二氧化碳培养箱中进行培养。8小时后，将样品取出进行多模态成像。
将经过培养的样品连同培养皿一起置于载物台3中央，载物台3中央有一圆孔，且 由于培养皿是透明的，因此透射光、反射光均可直接照射至光子晶体水凝胶薄膜上。如图3所示为受细胞牵引力作用下的光子晶体水凝胶薄膜2的反射光作用示意图，光子晶体水凝胶薄膜2在待测细胞作用下发生形变而使入射的反射光方向变化，进而成像组件获得表征细胞牵引力的阴影图像。成纤维细胞在光子晶体薄膜上生长会对光子晶体薄膜施加牵引力，使得光子晶体膜弯曲形成褶皱或突起。光子晶体对光线的反射除了具有波长选择性，还有镜面的特性，即入射光与反射光与该点的法线呈对称分布。因此，细胞施加给光子晶体水凝胶薄膜2的牵引力越大，薄膜与初始平行位置形成的倾角越大，便会有更多的光线反射到物镜以外的方向，在相机拍摄的图案中也就越暗。
将上述细胞培养皿取出置于载物台3上后，并使光子晶体水凝胶薄膜2垂直于主光轴放置在载物台3上。这样可以使透射光和反射光很好地在光子晶体水凝胶薄膜上进行透射、反射，使得到的图像质量更优。将分束镜配置为可见光半透半反。开卤钨灯荧光激发光源作为反射光源，打开荧光光闸。打开相差光源作为透射光源，调整相差光源和荧光激发光源的亮度。使用10x物镜，数值孔径为0.45，对Z轴进行调节，聚焦到聚丙烯酰胺光子晶体水凝胶薄膜上。关闭透射光源，使用显微镜的相机以800*600分辨率对需要研究的区域进行拍照，得到力学模态信息。关闭荧光光闸，即关闭反射光源。打开透射光源，使用显微镜的相机以800*600分辨率对需要研究的区域进行拍照，得到普通相差模态信息。
使用中值滤波对力学模态照片进行预处理，中值滤波模板大小为10*10。由于光线明暗变化的规律与Lambert模型类似，因此可使用Emmanuel Prados在DOI:10.1007/s10851-006-6899-x论文中提到的图像处理方法，对图像进行处理，得到光子晶体薄膜表面的三维形貌信息，反映了细胞对基底作用的力的大小。这样，结合前面拍得的相差图像，便得到了该区域的细胞形态和力学的多模态信息，进而得到待测细胞的牵引力分布。
将新生小鼠心肌原代成纤维细胞在聚丙烯酰胺光子晶体水凝胶薄膜上培养后使用胰酶消化，如图4-1、4-2、4-3分别是胰酶加入30s时的力学模态成像图片、力学模态和相差模态共成像图片以及根据力学模态信息重建出的薄膜表面形貌图；如图5-1、5-2、5-3分别是胰酶加入60s时的力学模态成像图片、力学模态和相差模态共成像图片以及根据力学模态信息重建出的薄膜表面形貌图；如图6-1、6-2、6-3分别是胰酶加入90s时的力学模态成像图片、力学模态和相差模态共成像图片以及根据力学模态信息重建出的薄膜表面形貌图。
实施例2：
本实施例中显微镜的结构与实施例1相同，其中光子晶体水凝胶薄膜选用杨氏模量为30kPa，中心反射波长位于600nm，反射峰半峰宽为20nm，最强反射波段反射率40％， 非禁带范围透光率大于85％，厚度为20微米，直径为1厘米的聚丙烯酰胺光子晶体水凝胶薄膜固定在支架上，并将聚丙烯酰胺光子晶体水凝胶薄膜连同支架一起放在容器中。
使用Sulfo-SANPAH活化光子晶体水凝胶表面的氨基后使用HEPES缓冲液清洗三次后浸泡在HEPES缓冲的0.01mg/mL的购置于东南大学苏州医疗器械研究院的心肌外基质提取物蛋白溶液中，4摄氏度过夜。将前述光子晶体水凝胶薄膜浸泡在α-MEM培养基中过夜后移到细胞培养皿中，再快速加入10％胎牛血清的α-MEM培养基浸没光子晶体水凝胶。加入新生SD大鼠心脏原代心肌细胞在10％胎牛血清的α-MEM培养基中的分散液，置于二氧化碳培养箱中进行培养。48小时后，将样品取出进行多模态成像。
将培养后的细胞培养皿取出后置于显微镜的载物台3上，分束镜配置为可见光半透半反。打开倒置显微镜卤钨灯荧光激发光源作为反射光源，打开荧光光闸。打开相差光源作为透射光源，调整相差光源和荧光激发光源的亮度。使用20x的物镜，数值孔径为0.45，对Z轴进行调节，聚焦到光子晶体薄膜上。关闭透射光源，使用显微镜的相机以800*600分辨率对需要研究的区域以30帧的帧率进行录像，得到力学模态信息。关闭荧光光闸，即关闭反射光源。打开透射光源，使用显微镜的相机以800*600分辨率对需要研究的区域以30帧的帧率进行录像，得到普通相差模态信息。
将得到的力学模态录像逐帧提取得到力学模态图像。使用中值滤波对力学模态照片进行预处理，中值滤波模板大小为10*10。使用Emmanuel Prados在DOI:10.1007/s10851-006-6899-x论文中提到的处理方法，对图像进行处理，得到光子晶体薄膜表面的三维形貌信息，反映了细胞对基底作用的力的大小。由于反射光的强度由光子晶体水凝胶薄膜的反射率和反射光源的光强决定，又由于我们所使用的光子晶体水凝胶在最强反射峰位置的反射率高达60％，因此可以使用传统的CMOS或CCD相机连续的力学模态信息。

Claims (7)

  1. 一种多模态生物力学显微镜，其特征在于：包括从上至下依次设置的透射光源(1)、光子晶体水凝胶薄膜(2)、载物台(3)、反射光源(4)、成像组件；所述透射光源(1)用于待测细胞形态成像；所述光子晶体水凝胶薄膜(2)用作待测细胞培养的基底和测量细胞牵引力的传感器；所述反射光源(4)用于测量待测细胞力学信息，并且反射光源(4)的光谱包含光子晶体水凝胶薄膜(2)的反射峰带宽范围；
    所述光子晶体水凝胶薄膜(2)置于载物台(3)上并保持悬空状态，使得透射光可以穿透薄膜到达成像组件，反射光经所述薄膜反射后到达成像组件；当待测细胞置于光子晶体水凝胶薄膜(2)上后，支撑所述待测细胞的光子晶体水凝胶薄膜(2)发生形变，使得光子晶体水凝胶薄膜(2)上的反射光方向发生改变，所述成像组件收集反射光和透射光并进行成像，得到表征细胞牵引力的阴影图像。
  2. 根据权利要求1所述的多模态生物力学显微镜，其特征在于：所述光子晶体水凝胶薄膜(2)在450-800nm波段内的最强反射波段反射率大于35％，非禁带范围透光率大于80％。
  3. 根据权利要求1所述的多模态生物力学显微镜，其特征在于：所述光子晶体水凝胶薄膜(2)的厚度为5-200微米。
  4. 根据权利要求1所述的多模态生物力学显微镜，其特征在于：所述光子晶体水凝胶薄膜(2)的杨氏模量为1-100kPa。
  5. 根据权利要求1所述的多模态生物力学显微镜，其特征在于：所述光子晶体水凝胶薄膜(2)的最强反射波长为450-800纳米。
  6. 根据权利要求1所述的多模态生物力学显微镜，其特征在于：所述显微镜还包括物镜(6)，所述物镜(6)的数值孔径为0.1-0.9，放大倍率为2-60倍。
  7. 一种基于权利要求1所述的显微镜的多模态生物力学测量方法，其特征在于：包括以下步骤：
    (1)将光子晶体水凝胶薄膜(2)进行前处理，后将待测细胞置于光子晶体水凝胶薄膜(2)上进行培养；
    (2)将步骤(1)得到的样品置于载物台(3)上，光子晶体水凝胶薄膜(2)在待测细胞作用下发生形变而使入射的反射光方向变化，进而获得表征细胞牵引力的阴影图像；
    (3)根据阴影图像通过算法得到待测细胞的牵引力分布。
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