WO2022052361A1 - 光子晶体显微镜和细胞力学测量方法 - Google Patents

光子晶体显微镜和细胞力学测量方法 Download PDF

Info

Publication number
WO2022052361A1
WO2022052361A1 PCT/CN2020/137819 CN2020137819W WO2022052361A1 WO 2022052361 A1 WO2022052361 A1 WO 2022052361A1 CN 2020137819 W CN2020137819 W CN 2020137819W WO 2022052361 A1 WO2022052361 A1 WO 2022052361A1
Authority
WO
WIPO (PCT)
Prior art keywords
photonic crystal
crystal substrate
imaging
substrate
detection light
Prior art date
Application number
PCT/CN2020/137819
Other languages
English (en)
French (fr)
Inventor
顾忠泽
李奇维
陈早早
Original Assignee
东南大学苏州医疗器械研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 东南大学苏州医疗器械研究院 filed Critical 东南大学苏州医疗器械研究院
Priority to EP20953146.6A priority Critical patent/EP4212857A1/en
Publication of WO2022052361A1 publication Critical patent/WO2022052361A1/zh
Priority to US18/180,120 priority patent/US20230221538A1/en

Links

Images

Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G01N21/84—Systems specially adapted for particular applications
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B1/00—Optical elements characterised by the material of which they are made; Optical coatings for optical elements
    • G02B1/002—Optical elements characterised by the material of which they are made; Optical coatings for optical elements made of materials engineered to provide properties not available in nature, e.g. metamaterials
    • G02B1/005—Optical elements characterised by the material of which they are made; Optical coatings for optical elements made of materials engineered to provide properties not available in nature, e.g. metamaterials made of photonic crystals or photonic band gap materials
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
    • G02B26/007—Optical devices or arrangements for the control of light using movable or deformable optical elements the movable or deformable optical element controlling the colour, i.e. a spectral characteristic, of the light

Definitions

  • the present disclosure belongs to the technical field of crystal microscopes, and in particular relates to a photonic crystal microscope and a cell mechanics measurement method.
  • the mechanical properties of living materials can actively change the response to perturbation, resulting in more or less compaction of the tissue under constant force.
  • the timescales of these deformation assays do not allow the measurement of force fluctuations, making it impossible to perform important studies of rapidly contracting cells such as myocytes.
  • Another method was used to measure the forces developed in compacted hydrogels. The first method is to use a gel large enough to connect to an external isoforce transducer. These sensors are off-the-shelf devices and force changes their voltage or resistance. Therefore, force, rather than displacement, is measured directly from contracting tissue. This system has been used to measure the forces generated by cells from highly contractile tissues, including skin fibroblasts, cardiomyocytes, and skeletal muscle cells.
  • the second approach involves adding a cantilever of known stiffness to the system so that when the tissue contracts, the cantilever flexes.
  • the displacement of the free end of the cantilever can be detected with an optical microscope and the tissue contractile force can be theoretically calculated from the displacement.
  • An advantage of this system is that the deformation of many cantilevers can be quickly measured.
  • the systems can also be more compact than the electronic detection systems described above, meaning they require fewer cells and less extracellular matrix material and do not require manual mounting onto a single sensor.
  • vertical cantilevers have been able to create systems from silicon elastomers that can measure the resultant force of forces as low as 100-600 cells. These systems have become increasingly important tools for measuring forces in cells, such as cardiomyocytes.
  • TFM Cellular traction force microscopy
  • the algorithm determines the displacement of the beads and the force required by the cells by analyzing the two images (or sequences of images) produced. Since beads are much smaller than cells, TFM allows cellular forces to be mapped with subcellular resolution. This method has been applied to the measurement of various cell biological processes, such as the characterization of the mechanodynamics of adhesion maturation, migration, differentiation and malignant transformation. The computational analysis required to calculate bead displacement and force has been a significant hurdle, and TFM calculations are complex, subtle, and difficult to verify. This is partly due to small errors in measuring the position of the microbeads that can cause larger errors in force calculations and mathematical ill-posedness.
  • the size and spacing of the microbead markers and the optical resolution of the microscope determine the spatial resolution of the observed deformation field, which, in turn, determines the mechanical resolution. Therefore, drawing high-resolution traction maps requires high-resolution imaging, which greatly limits the application of this method. The high magnification objectives required for high resolution also limit throughput. High-dose phototoxicity will also cause greater interference to the test sample itself.
  • the present disclosure aims to solve at least one of the technical problems existing in the prior art, and provides a photonic crystal microscope and a cell mechanics measurement method.
  • the photonic crystal microscope includes a photonic crystal substrate, a stage, a detection light source and an imaging assembly, the photonic crystal substrate is above the stage, and the detection light source and the imaging assembly is sequentially located on the side of the stage away from the photonic crystal substrate, the photonic crystal substrate is used for culturing the cells to be tested, and the cells to be tested grow on the photonic crystal substrate , the photonic crystal substrate can be deformed; wherein,
  • the detection light source used to emit detection light to the photonic crystal substrate
  • the photonic crystal substrate for reflecting the probe light to the imaging component
  • the imaging component is configured to receive the reflected light from the photonic crystal substrate for imaging, so as to obtain the force information between the cell to be tested and the photonic crystal substrate by using the imaging pattern.
  • the photonic crystal substrate comprises:
  • a photonic crystal film, the photonic crystal film is arranged on the side of the support layer away from the stage.
  • the reflectivity of the photonic crystal thin film in the strongest reflection band in the wavelength band of 430nm-700nm is greater than 35%, and the transmittance in the non-forbidden band range is greater than 70%.
  • the thickness of the photonic crystal thin film ranges from 20 ⁇ m to 80 ⁇ m.
  • the Young's modulus of the photonic crystal thin film ranges from 0.5 kPa to 100 kPa.
  • the photonic band gap of the photonic crystal thin film ranges from 430 nm to 700 nm.
  • the photonic crystal microscope further includes an objective lens, and the objective lens is disposed between the object stage and the detection light source.
  • the numerical aperture of the objective lens ranges from 0.1 to 0.9, and/or the magnification of the objective lens ranges from 1 to 100 times.
  • the imaging assembly includes an optical filter and a photosensitive element, and the optical path of the optical filter is located between the photosensitive element and the stage.
  • Another aspect of the present disclosure provides a method for measuring cell mechanics, using the photonic crystal microscope described above, the method comprising:
  • the detection light source emits detection light to the photonic crystal substrate
  • the photonic crystal substrate reflects the probe light to the imaging assembly
  • the imaging component receives the reflected light from the photonic crystal substrate for imaging, so as to obtain the force information between the cell to be tested and the photonic crystal substrate by using the imaging pattern.
  • the photonic crystal microscope and the cell mechanics measurement method disclosed in the present disclosure propose a method for quantitatively measuring the biomechanics of cells by using a photonic crystal substrate, imaging the reflected light of the photonic crystal substrate, and using an algorithm to obtain the mechanical interaction information between cells and the substrate ; Under the premise of ensuring the accuracy of subcellular measurement, the throughput has been greatly improved.
  • the optical characteristics of photonic crystals make the initial state stable and known during the measurement process, which greatly simplifies the complexity of the algorithm and the sensitivity to noise. and experimental complexity.
  • the uniformity of the photonic crystal substrate interferes with the physiological activities of the cells itself much less than the discrete micro-pillar array, which can achieve more accurate restoration of the physiological environment and obtain more accurate results.
  • the mechanical information is contained in the reflected light, and its brightness is usually much higher than that of fluorescence, which greatly reduces the influence of phototoxicity on the sample to be tested.
  • FIG. 1 is a schematic structural diagram of a photonic crystal microscope in an embodiment of the disclosure
  • FIG. 2 is a schematic diagram of the reflected light effect of the photonic crystal film not under the action of cell pulling force according to another embodiment of the present disclosure
  • FIG. 3 is a schematic diagram of the reflected light effect of the photonic crystal film under the action of cell traction in another embodiment of the present disclosure
  • FIG. 4 is an imaging diagram of a photonic crystal under a 10 ⁇ objective lens in another embodiment of the present disclosure.
  • FIG. 5 is a phase contrast modal diagram under a 10x objective lens in another embodiment of the present disclosure.
  • FIG. 6 is a mechanical heat map under a 10 ⁇ objective lens in another embodiment of the present disclosure.
  • a photonic crystal microscope 100 includes a photonic crystal substrate 110 , a stage 120 , a detection light source 130 and an imaging component 140 .
  • the photonic crystal substrate 110 is arranged on the stage 120 , and the photonic crystal substrate 110 is used for culturing the cells to be tested (not shown in the figure), and in the When the cells to be tested grow on the photonic crystal substrate 110, the cell growth area causes the photonic crystal substrate 110 to deform, that is, in the photonic crystal microscope 100, the photonic crystal substrate 110 is used for culturing the In addition to measuring cells, it is also used as a sensor for measuring cell traction.
  • the detection light source 130 and the imaging component 140 are sequentially located on the side of the stage 120 away from the photonic crystal substrate 110 , that is, the detection light source 130 is located on the object. Between stage 120 and imaging assembly 140 .
  • the detection light source 130 is used to emit detection light to the photonic crystal substrate 110, and the detection light source 130 is used to measure the mechanical information of the cell to be tested.
  • the above-mentioned photonic crystal substrate 110 is also used to reflect the probe light to the imaging component 140 .
  • the imaging component 140 is configured to receive the reflected light from the photonic crystal substrate 110 for imaging, so as to obtain the force information between the cell to be tested and the photonic crystal substrate by using the imaging pattern.
  • the photonic crystal substrate 110 is deformed, so that after receiving the detection light emitted by the detection light source 130 , the deformed photonic crystal substrate 110 can change the photonic forbidden band so that the wavelength component of the reflected light can be changed, as shown in FIG. 3 .
  • the imaging component 140 receives the reflected light and performs imaging to obtain the mechanical interaction information between the cells to be tested and the photonic crystal substrate 110 by using the imaging pattern.
  • the imaging pattern can be input to On terminal equipment such as computers, the terminal equipment analyzes and extracts the imaging graphics, and obtains the mechanical interaction information between the cells to be tested and the photonic crystal substrate through algorithm calculation.
  • FIG. 1 and FIG. 2 when the cells to be tested are not placed on the photonic crystal substrate 110 , the wavelength component of the reflected light of the photonic crystal substrate 110 does not change.
  • the photonic crystal microscope of the embodiment of the present disclosure proposes a method for quantitatively measuring the biomechanics of cells by using a photonic crystal substrate, imaging the reflected light of the photonic crystal substrate, and using an algorithm to obtain the mechanical interaction information between cells and the substrate; Under the premise of subcellular measurement accuracy, the throughput has been greatly improved.
  • the optical characteristics of photonic crystals make the initial state stable and known during the measurement process, which greatly simplifies the complexity of the algorithm and the sensitivity to noise. and experimental complexity.
  • the uniformity of the photonic crystal substrate interferes with the physiological activities of the cells itself much less than the discrete micro-pillar array, which can achieve more accurate restoration of the physiological environment and obtain more accurate results.
  • the mechanical information is contained in the reflected light, and its brightness is usually much higher than that of the fluorescence, so there is no need to increase the light intensity of the light source, which greatly reduces the phototoxicity to cells. sample impact.
  • the photonic crystal substrate 110 includes a support layer and a photonic crystal film, and the support layer is light-transmitting and disposed on the stage 120 .
  • the photonic crystal thin film is disposed on the side of the support layer away from the stage 120 .
  • the initial state of the composite material of the photonic crystal thin film serving as the sensing unit is known, and there is no need to construct a corresponding relationship, which can simplify the measurement process.
  • the reflectivity of the photonic crystal film in the strongest reflection band in the 430nm-700nm band is greater than 35%, and the light transmittance in the non-forbidden band range is greater than 70%.
  • the thickness of the photonic crystal thin film ranges from 20 ⁇ m to 80 ⁇ m.
  • the Young's modulus of the photonic crystal thin film ranges from 0.5kPa to 100kPa.
  • the photonic band gap of the photonic crystal thin film ranges from 430 nm to 700 nm.
  • the photonic crystal microscope 100 further includes an objective lens 150 disposed between the object stage 120 and the detection light source 130 .
  • the numerical aperture of the objective lens 150 is in the range of 0.1 to 0.9, and the magnification of the objective lens 150 is in the range of 1 to 100 times.
  • the numerical aperture and magnification of the objective lens 150 can also be selected from other values. value, which is not limited in this embodiment of the present disclosure.
  • the imaging assembly 140 includes an optical filter 141 and a photosensitive element 142 , and the optical path where the optical filter 141 is located is located between the photosensitive element 142 and the stage 120 .
  • the photonic crystal microscope 100 provided by the implementation of the present disclosure can acquire mechanical modal information.
  • the photonic crystal film is deformed by the force exerted by the cells, and the photonic forbidden band of the photonic crystal film at the corresponding position is changed, so that The photonic crystal reflectance spectrum changes.
  • the strain of the film by an algorithm and according to the mechanical and geometric properties of the material itself, the deformation and stress of the substrate perpendicular to the initial state caused by the cells can be obtained.
  • the measurement of cell traction force is based on the fact that the substrate deformation information caused by cells is given to reflected light according to the photonic crystal, and the cell is acquired in real time through the collection of reflected light and algorithm extraction, so that the strain stress and deformation, flux and accuracy of the photonic crystal material are obtained in real time. Very high.
  • the region of interest is repeatedly imaged before the cells attach until the end of the experiment to determine the trajectory of the beads in response to local stress changes. If you remove it and then return to the field of view, it may be impossible 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 perform fluorescence imaging on the sample of interest to determine the bead at the current moment. After positioning, trypsin is added to allow separation between the cells and the substrate.
  • the imaging is continued, and the movement trajectory of the microbeads in the process of the substrate from the state of uneven stress distribution to the state of no stress is obtained, so as to obtain the corresponding relationship of the microbeads between the two states.
  • the photonic crystal microscope provided by the present invention has no such limitation, because the initial state of the photonic crystal hydrogel film as the sensing unit is known, and there is no need to construct the corresponding relationship.
  • Cell traction force microscopes face rapidly changing samples, such as beating myocardium. Due to the weak fluorescence of fluorescent microbeads, high-energy excitation light and highly sensitive microscope cameras are required to obtain sufficiently high temporal resolution, which may cause damage to the samples. The damage also has higher requirements on the hardware of the experiment.
  • the mechanical information in the photonic crystal microscope provided by the present disclosure is contained in the reflection spectrum, 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, thereby greatly reducing the effect of phototoxicity on cell samples.
  • Another aspect of the present disclosure provides a method for measuring cell mechanics.
  • the photonic crystal microscope described above is used.
  • the method includes:
  • the detection light source emits detection light to the photonic crystal substrate
  • the photonic crystal substrate reflects the probe light to the imaging assembly
  • the imaging component receives the reflected light from the photonic crystal substrate for imaging, so as to obtain the force information between the cell to be tested and the photonic crystal substrate by using the imaging pattern.
  • the photonic crystal measurement method of the embodiment of the present disclosure proposes a method for quantitatively measuring the biomechanics of cells by using a photonic crystal substrate, imaging the reflected light of the photonic crystal substrate, and using an algorithm to obtain mechanical interaction information between cells and the substrate; Under the premise of ensuring the accuracy of subcellular measurement, the throughput has been greatly improved.
  • the optical characteristics of photonic crystals make the initial state stable and known during the measurement process, which greatly simplifies the complexity of the algorithm and the sensitivity to noise. and experimental complexity.
  • the uniformity of the photonic crystal substrate interferes with the physiological activities of the cells itself much less than the discrete micro-pillar array, which can achieve more accurate restoration of the physiological environment and obtain more accurate results.
  • the mechanical information is contained in the reflected light, and its brightness is usually much higher than that of the fluorescence, so there is no need to increase the light intensity of the light source, and the biological sample is better maintained. the original state.
  • the photonic crystal substrate 110 may be a photonic crystal hydrogel composed of polyacrylamide and silicon dioxide with a thickness of about 30 ⁇ m attached to the upper surface of the quartz plate, wherein the polyacrylamide photonic crystal hydrogel
  • the Young's modulus of the film is 3kPa; the central reflection wavelength of the photonic crystal film is located at 550nm to obtain a larger measurement range; the half-peak width of the reflection peak is 20nm, the reflectivity of the strongest reflection band is 40%, and the non-bandgap range transmits light ratio is greater than 85% to obtain a higher signal-to-noise ratio.
  • the aforementioned photonic crystal hydrogel film was soaked in ⁇ -MEM medium overnight and then transferred to a cell culture dish, and then ⁇ -MEM medium with 10% fetal bovine serum was quickly added to immerse the photonic crystal hydrogel.
  • a dispersion of neonatal SD rat cardiac primary cardiomyocytes in ⁇ -MEM medium with 10% fetal bovine serum was added, and cultured in a carbon dioxide incubator. After 3 hours, the samples were imaged.
  • the cultured sample is placed in the center of the stage 120 together with the petri dish, and there is a circular hole in the center of the stage 120 .
  • 3 is a schematic diagram of the reflected light effect of the photonic crystal film under the action of the cell force.
  • the photonic crystal film on the photonic crystal substrate 110 is deformed under the action of the force exerted by the cell to be tested, so that the reflected wavelength component of the detection light is change.
  • the reflected light is collected by the imaging component and then algorithmically obtains information on the mechanical interaction of the cells with the substrate.
  • the growth of cardiomyocytes on the photonic crystal film exerts a force on the photonic crystal film, causing the photonic crystal film to deform.
  • the reflection of light by photonic crystals has wavelength selectivity, that is, the reflection spectrum corresponds to the photon forbidden band.
  • the reflection spectrum corresponds to the photon forbidden band.
  • the photonic crystal substrate 110 is placed on the stage 120 perpendicular to the main optical axis.
  • the transmitted light and the reflected light can be well transmitted and reflected on the photonic crystal film, so that the obtained image quality is better.
  • Closing the fluorescence shutter cuts off the reflected light source.
  • Turn on the transmitted light source, and use the microscope camera to take pictures of the area to be studied with a resolution of 4800*3600 to obtain common phase contrast modal information.
  • the mechanical modal photos are preprocessed using median filtering, and the median filtering template size is 5*5. Since the wavelength shift percentage of the photonic crystal reflection peak position and the strain are approximately linear, the hue of each pixel of the image can be converted into the reflection peak position information through the calibrated camera hue relationship, and then combined with the initial peak position information to obtain the corresponding position strain, and finally according to The Young's modulus of the material and the strain at each point obtain the stress distribution in the cell area to be measured.
  • the method for obtaining the relationship between the hue of the color picture captured by the camera and the peak position relationship of the photonic crystal reflected light is obtained. Then, the peak position of the spectrum at the same time is corresponding to the hue of the photo, and the mapping relationship between the photonic crystal reflection peak position and the hue is obtained.
  • the primary myocardial cells of neonatal mouse myocardium were cultured on polyacrylamide photonic crystal hydrogel film, and the conventional phase contrast photos were obtained using the phase contrast mode, and the mechanical mode map was obtained using the reflection mode. Strain information is obtained from the mapping relationship of the band gap peak positions.
  • Figure 4, Figure 5, and Figure 6 are the photonic crystal imaging images, phase contrast modal images, and calculated stress heat maps obtained by direct shooting, respectively. From the figure, the local strain caused by the interaction between subcellular structures (eg, lamellipodia) and the substrate can be clearly seen, and multiple cells can be observed simultaneously in a millimeter-scale field of view, achieving high throughput and high precision Measurement.
  • subcellular structures eg, lamellipodia

Landscapes

  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Optics & Photonics (AREA)
  • Analytical Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Health & Medical Sciences (AREA)
  • Astronomy & Astrophysics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

一种光子晶体显微镜(100)和细胞力学测量方法。光子晶体显微镜(100)包括光子晶体基底(110)、载物台(120)、探测光源(130)和成像组件(140)，光子晶体基底(110)在载物台(120)上方，探测光源(130)和成像组件(140)依次位于载物台(120)背离光子晶体基底(110)的一侧，光子晶体基底(110)用于培养待测细胞，并且，在待测细胞在光子晶体基底(110)上生长时，光子晶体基底(110)能够产生形变；其中，探测光源(130)，用于向光子晶体基底(110)发出探测光；光子晶体基底(110)反射探测光至成像组件(140)；成像组件(140)，接收来自光子晶体基底(110)的反射光进行成像，以利用成像图形得到待测细胞与光子晶体基底(110)之间的作用力信息。在保证亚细胞测量精度的前提下实现了通量的大幅度提高，简化了算法的复杂度、提高了时间分辨率、降低装置成本与实验的复杂度。

Description

光子晶体显微镜和细胞力学测量方法 技术领域
本公开属于晶体显微镜技术领域，具体涉及一种光子晶体显微镜和细胞力学测量方法。
背景技术
细胞产生的力是细胞粘附、信号传递和功能的关键调节器，它们也是发育中形态发生事件的重要驱动力。在过去的数十年中，已经开发了多种方法来测量这些力。虽然广大研究人员对了解这些力在生物学中的贡献有很大的兴趣，但对他们进行广泛的测量仍然具有挑战性。
表征细胞力存在的最简单方法涉及测量细胞、基质或组织的变形，而不试图将这些变形与实际的力联系起来。例如，嵌入在胶原蛋白凝胶中的细胞将压缩凝胶，可模仿在伤口愈合期间发生的收缩。通过在孔板中聚合的凝胶的直径变化来初步判断细胞力的大小。这种方法的优点是，不需要复杂的理论或测量方法对被变形的材料的机械变形进行复杂计算，以转换变形为力。然而，基于变形的方法也有缺点。隐含在分析中的假设是，更多的压实或回缩意味着更多的细胞力，但断裂，塑性和材料的粘弹性可能会使这个假设无效。此外，活体材料的力学性能可以主动改变对扰动的反应，导致组织在恒定的力下发生或多或少的压实。此外，这些变形测定的时间尺度不允许力波动的测量，无法进行重要的快速收缩细胞，如肌细胞的研究。另一种方法被用来测量压实水凝胶中产生的力。第一种方法是使用足够大的凝胶，以连接到外部等力传感器。这些传感器是现成的设备，力会改变其电压或电阻。因此，力，而不是位移，是直接从收缩组织测量。这种系统已被用于测量由细胞产生的力从高度收缩的组织，包括皮肤成纤 维细胞，心肌细胞和骨骼肌细胞。虽然这些系统提供了连续和长期的组织收缩力的测量，所需的信号处理，从力传感器的电信号输出转换为实际的力所需的技能超出了大多数标准生物实验室的能力范围。此外，这些方法是有限的吞吐量，因为传感器的工作范围的下限通常是在微到毫微牛顿需要使用大型设备并手动安装到力传感器。
第二种方法是在系统中加入已知刚度的悬臂，这样当组织收缩时，悬臂就会弯曲。悬臂自由端的位移可以用光学显微镜检测并通过位移来理论计算组织收缩力。该系统的一个优点是可以快速测量许多悬臂的变形。该系统也可以比上述电子检测系统具有更紧凑，这意味着它们仅需要更少的细胞和更少的细胞外基质材料且不需要手动安装到单个传感器上。最近，垂直悬臂已经可以从硅弹性体创建的系统，可以测量力低至100-600个细胞的合力。这些系统已经成为越来越重要的工具，用于测量细胞的力，如心肌细胞。虽然使用这些微制造的构造测量力只需要一个具有适当长工作距离的显微镜但系统的制造同样需要生物实验室中的非标技术。悬臂是由软光刻复制成型。然而，创建原始硅母片需要微细加工设施。虽然代工厂会以一定的成本制造硅母片，但需要指定专业的技术设计。测量组织构建产生的净收缩力可以提供有关驱动组织变形的信号的定量信息，特别是ECM的作用。然而，ECM重塑和细胞力是耦合在所产生的集合测量，因此，这取决于用于生成含细胞的ECM凝胶的具体配方。这些因素使得很难比较不同研究之间的测量结果，也很难分离出单个细胞产生的力量。
细胞牵引力显微镜(TFM)通过跟踪合成的弹性聚合物基体的变形进行力学测量。这种方法，及其变体，是用于测量细胞力最广泛使用的技术。在标准的TFM中，小(≤1微米)荧光珠混合到硅胶或聚丙烯酰胺(PA)基质中，作为受托标记，可以在空间和时间上被光学显微镜跟踪。一个典型的TFM实验涉及以下内容：光学成像珠子在受压状态下的分布，通过细胞裂解、分离、或肌球蛋白抑制释放细胞牵引力；然后再次对荧光微珠成像，以确 定它们在非受压状态下的位置。算法是通过分析所产生的两个图像(或图像序列)来确定由细胞引起的珠子的位移和所需的力。由于微珠远小于细胞，TFM允许细胞的力量被映射与亚细胞分辨率。该方法已经应用于各种细胞生物学过程的测量，如粘附成熟、迁移、分化和恶性转化的力动力学的表征。计算微珠位移和力所需的计算分析一直是一个重大的障碍，TFM的计算是复杂、细微和难以验证。这在一定程度上是由于测量微珠位置的微小误差可以引起较大误差的力计算和数学上的非适定性。此外，微珠标记的大小和间距和显微镜的光学分辨率决定了观察到的变形场的空间分辨率，反过来，决定了力学分辨率。因此，绘制高分辨率的牵引力图需要高分辨率的成像，极大的限制了该方法的应用。高分辨需要的高倍物镜还使得通量受到限制。高剂量的光毒性，也会对待测样本本身造成较大的干扰。
尽管基于微柱阵列的方法和基于荧光共振能量转移的方法得到了长足的发展，但仍然存在制备困难和测量设备要求高等局限。此外，目前的方法仅局限于宏观组织或微观的数个细胞的力学测量，无法进行毫米视野范围内亚细胞分辨率的力学测量。
发明内容
本公开旨在至少解决现有技术中存在的技术问题之一，提供一种光子晶体显微镜和细胞力学测量方法。
本公开的一个方面，提供一种光子晶体显微镜，所述光子晶体显微镜包括光子晶体基底、载物台、探测光源和成像组件，所述光子晶体基底在所述载物台上方，所述探测光源和所述成像组件依次位于所述载物台背离所述光子晶体基底的一侧，所述光子晶体基底用于培养待测细胞，并且，在所述待测细胞在所述光子晶体基底上生长时，所述光子晶体基底能够产生形变；其中，
所述探测光源，用于向所述光子晶体基底发出探测光；
所述光子晶体基底，用于反射所述探测光至所述成像组件；
所述成像组件，用于接收来自所述光子晶体基底的反射光进 行成像，以利用成像图形得到所述待测细胞与所述光子晶体基底之间的作用力信息。
在一些可选地实施方式中，所述光子晶体基底包括：
透光的支撑层；
光子晶体薄膜，所述光子晶体薄膜设置在所述支撑层背离所述载物台的一侧。
在一些可选地实施方式中，所述光子晶体薄膜在430nm～700nm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70％。
在一些可选地实施方式中，所述光子晶体薄膜的厚度范围为20μm～80μm。
在一些可选地实施方式中，所述光子晶体薄膜的杨氏模量范围为0.5kPa～100kPa。
在一些可选地实施方式中，所述光子晶体薄膜的光子禁带范围为430nm～700nm。
在一些可选地实施方式中，所述光子晶体显微镜还包括物镜，所述物镜设置在所述载物台和所述探测光源之间。
在一些可选地实施方式中，所述物镜的数值孔径范围为0.1～0.9，和/或，所述物镜的放大倍率范围为1倍～100倍。
在一些可选地实施方式中，所述成像组件包括滤光器和感光元件，所述滤光器所在光路位于所述感光元件和所述载物台之间。
本公开的另一方面，提供一种细胞力学测量方法，采用前文记载的所述的光子晶体显微镜，所述方法包括：
将所述光子晶体基底置于所述载物台上，观察所述光子晶体基底在待测细胞作用下发生形变；
所述探测光源向所述光子晶体基底发出探测光；
所述光子晶体基底反射所述探测光至所述成像组件；
所述成像组件接收来自所述光子晶体基底的反射光进行成像，以利用成像图形得到所述待测细胞与所述光子晶体基底之间的作用力信息。
本公开的光子晶体显微镜和细胞力学测量方法，提出了使用光子晶体基底对细胞的生物力学进行定量测量的方法，对光子晶体基底的反射光进行成像，使用算法获得细胞与基底的力学相互作用信息；在保证亚细胞测量精度的前提下实现了通量的大幅度提高。此外，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。另外，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。最后，本公开实施例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，极大的降低了光毒性对待测样本的影响。
附图说明
图1为本公开一实施例中光子晶体显微镜的结构示意图；
图2为本公开另一实施例中不受细胞牵引力作用下的光子晶体薄膜的反射光作用示意图；
图3为本公开另一实施例中受细胞牵引力作用下的光子晶体薄膜的反射光作用示意图；
图4为本公开另一实施例中在10x物镜下，光子晶体成像图；
图5为本公开另一实施例中在10x物镜下，相差模态图；
图6为本公开另一实施例中在10x物镜下，力学热图。
具体实施方式
为使本领域技术人员更好地理解本公开的技术方案，下面结合附图和具体实施方式对本公开作进一步详细描述。
如图1所示，一种光子晶体显微镜100，所述光子晶体显微镜100包括光子晶体基底110、载物台120、探测光源130和成像组件140。
示例性的，如图1所示，所述光子晶体基底110设置在所述载物台120上，所述光子晶体基底110用于培养待测细胞(图中并未示出)，并且，在所述待测细胞在所述光子晶体基底110上生长时，细胞生长区域使得光子晶体基底110产生形变，也就是说，在该光子晶体显微镜100中，光子晶体基底110除了用作培养所述待测细胞以外，还用作测量细胞牵引力的传感器。
示例性的，如图1所示，所述探测光源130和所述成像组件140依次位于所述载物台120背离所述光子晶体基底110的一侧，也就是说，探测光源130位于载物台120和成像组件140之间。探测光源130用于向所述光子晶体基底110发出探测光，该探测光源130用于测量待测细胞的力学信息。上述的光子晶体基底110还用于反射所述探测光至所述成像组件140。所述成像组件140，用于接收来自所述光子晶体基底110的反射光进行成像，以利用成像图形得到所述待测细胞与所述光子晶体基底之间的作用力信息。
具体地，如图1所示，在将所述待测细胞放置在所述光子晶体基底110上进行培养后，光子晶体基底110发生形变，这样，在接收到探测光源130所发出的探测光后，发生形变的光子晶体基底110可以使得光子禁带发生变化从而可以改变反射光的波长分量，如图3所示。如此，所述成像组件140在接收到反射光并进行成像，以利用成像图形得到所述待测细胞和所述光子晶体基底110之间的力学相互作用信息，例如，可以将该成像图形输入至电脑等终端设备上，该终端设备对成像图形进行分析和提取，经过算法计算得到待测细胞和光子晶体基底之间的力学相互作用信息。相反，如图1和图2所述，在光子晶体基底110上并未放置待测细胞时，光子晶体基底110的反射光的波长分量并不会发生改变。
本公开实施例的光子晶体显微镜，提出了使用光子晶体基底对细胞的生物力学进行定量测量的方法，对光子晶体基底的反射光进行成像，使用算法获得细胞与基底的力学相互作用信息；在 保证亚细胞测量精度的前提下实现了通量的大幅度提高。此外，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。另外，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。最后，本公开实施例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，大大降低了光毒性对细胞样品的影响。
示例性的，所述光子晶体基底110包括支撑层和光子晶体薄膜，所述支撑层透光且设置在所述载物台120上。所述光子晶体薄膜设置在所述支撑层背离所述载物台120的一侧。
本公开实施例的光子晶体显微镜，作为传感单元的光子晶体薄膜的复合材料的初始状态是已知的，不需要进行对应关系的构建，可以简化测量过程。
示例性的，所述光子晶体薄膜在430nm～700nm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70％。所述光子晶体薄膜的厚度范围为20μm～80μm。所述光子晶体薄膜的杨氏模量范围为0.5kPa～100kPa。所述光子晶体薄膜的光子禁带范围为430nm～700nm。当然，除了上述所列举的各项范围以外，本领域技术人员还可以根据实际需要，选择其他一些取值范围，本公开实施例对此并不限制。
示例性的，如图1所示，所述光子晶体显微镜100还包括物镜150，所述物镜150设置在所述载物台120和所述探测光源130之间。所述物镜150的数值孔径范围为0.1～0.9，所述物镜150的放大倍率范围为1倍～100倍，当然，除此以外，所述物镜150的数值孔径以及放大倍率还可以选择其他一些取值，本公开实施例对此并不限制。
示例性的，如图1所示，所述成像组件140包括滤光器141 和感光元件142，所述滤光器141所在光路位于所述感光元件142和所述载物台120之间。
下文将对本公开实施例的光子晶体显微镜的实现原理进行更为详细的说明：
如图1所示，本公开实施提供的光子晶体显微镜100，可进行力学模态信息获取。将待测细胞置于本公开实施例中光子晶体显微镜100的光子晶体基底110上进行培养后，光子晶体薄膜受到细胞施加的力而形变，改变了对应位置的光子晶体薄膜的光子禁带，使得光子晶体反射光谱发生变化。进一步，通过算法还原薄膜应变以及根据材料本身的力学和几何性质，可以得到细胞引起的垂直于初始状态的基底的形变和应力的大小。本公开实施例中细胞牵引力的测量是根据光子晶体将细胞引起的基底形变信息赋予反射光，通过反射光的采集和算法提取实时获取细胞使得光子晶体材料的应变应力和形变，通量和准确度极高。
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。而本发明提供的光子晶体显微镜进行测试，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。而本公开提供的光子晶体显微镜中的力学信息是蕴含在反射光谱中，其亮度通常远高于荧光，因此不需要额外增加光源光强，从而大大降低了光毒性对细胞样本的影响。
本公开的另一方面，提供一种细胞力学测量方法，采用前文记载的所述的光子晶体显微镜，光子晶体显微镜的具体结构可以参考前文相关记载，在此不作赘述。所述方法包括：
将所述光子晶体基底置于所述载物台上，观察所述光子晶体基底在待测细胞作用下发生形变；
所述探测光源向所述光子晶体基底发出探测光；
所述光子晶体基底反射所述探测光至所述成像组件；
所述成像组件接收来自所述光子晶体基底的反射光进行成像，以利用成像图形得到所述待测细胞与所述光子晶体基底之间的作用力信息。
本公开实施例的光子晶体测量方法，提出了使用光子晶体基底对细胞的生物力学进行定量测量的方法，对光子晶体基底的反射光进行成像，使用算法获得细胞与基底的力学相互作用信息；在保证亚细胞测量精度的前提下实现了通量的大幅度提高。此外，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。另外，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。最后，本公开实施例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，更好地维持了生物样本的原始状态。
具体地，可以一并结合图1，光子晶体基底110可以是在石 英片的上表面附着约30μm厚聚丙烯酰胺和二氧化硅构成的光子晶体水凝胶，其中聚丙烯酰胺光子晶体水凝胶薄膜的杨氏模量为3kPa；光子晶体薄膜的中心反射波长位于550nm，以获得较大的测量范围；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0.1mg/mL的鼠尾一型胶原蛋白溶液中，4摄氏度过夜，实现功能化修饰。
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。3小时后，将样品进行成像。
将经过培养的样品连同培养皿一起置于载物台120中央，载物台120中央有一圆孔。如图3所示为受细胞力作用下的光子晶体薄膜的反射光作用示意图，光子晶体基底110上的光子晶体薄膜在待测细胞施加的力的作用下发生形变而使探测光的反射波长分量发生变化。反射光被成像组件收集后通过算法获得细胞与基底的力学相互作用的信息。心肌细胞在光子晶体薄膜上生长会对光子晶体薄膜施加力，使得光子晶体膜发生形变。光子晶体对光线的反射具有波长选择性，即反射光谱与光子禁带相对应。因此，细胞给光子晶体薄膜施加朝向基底压力的点，反射光谱蓝移；细胞施加背向基底拉力的点，反射光谱红移，从而在相机中形成图案。
将上述细胞培养皿取出置于载物台120上后，并使光子晶体基底110垂直于主光轴放置在载物台120上。这样可以使透射光和反射光很好地在光子晶体薄膜上进行透射、反射，使得到的图像质量更优。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4800*3600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，即切断反射光源。打开透射光源，使用显微镜的相机以4800*3600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5*5。由于光子晶体反射光峰位置波长位移百分比和应变近似线性，因此可通过校准后的相机色相关系将图像各像素的色相转变成反射峰位置信息后结合初始峰位置信息得到对应位置的应变，最终根据材料的杨氏模量和各点的应变得到待测细胞区域的应力分布。
相机拍摄得到的彩色图片的色相和光子晶体反射光的峰位置关系的获得方法具体来说，使用纳米精度的压台对光子晶体薄膜进行挤压，使用光谱仪和相机同时记录光谱和彩色照片数据，再将同一时刻光谱的峰值位置和照片的色相进行对应，得到光子晶体反射峰位置和色相的映射关系。
将新生小鼠心肌原代心肌细胞在聚丙烯酰胺光子晶体水凝胶薄膜上培养后培养，使用相差模式获得常规的相差照片，使用反射模式获得力学模态图，提取色相信息后根据色相与光子禁带峰位置的映射关系得到应变信息。如图4、图5和图6分别是直接拍摄得到的光子晶体成像图、相差模态图、以及计算得到的应力热图。从图中既可以清晰的看到亚细胞结构(如，板状伪足)和基底的相互作用引起的局部应变，又能同时在毫米级视野观察多个细胞，实现了高通量与高精度的测量。
可以理解的是，以上实施方式仅仅是为了说明本公开的原理而采用的示例性实施方式，然而本公开并不局限于此。对于本领域内的普通技术人员而言，在不脱离本公开的精神和实质的情况下，可以做出各种变型和改进，这些变型和改进也视为本公开的保护范围。

Claims (9)

  1. 一种光子晶体显微镜，其特征在于，所述光子晶体显微镜包括光子晶体基底、载物台、探测光源和成像组件，所述光子晶体基底在所述载物台上方，所述探测光源和所述成像组件依次位于所述载物台背离所述光子晶体基底的一侧，所述光子晶体基底用于培养待测细胞；其中，
    所述探测光源，用于向所述光子晶体基底发出探测光；
    所述光子晶体基底，用于反射所述探测光至所述成像组件，所述光子晶体基底包括透光的支撑层和光子晶体薄膜，所述光子晶体薄膜设置在所述支撑层背离所述载物台的一侧，所述光子晶体薄膜非悬空地设置在所述载物台上方，在所述待测细胞在所述光子晶体薄膜上生长时，该待测细胞会对光子晶体薄膜施加作用力，使得所述光子晶体薄膜发生形变，非悬空设置的所述光子晶体薄膜对光线的反射具有波长选择性，以使得发生形变的所述光子晶体基底可以改变反射光的波长分量，也即反射光谱与光子禁带相对应，在所述待测细胞给光子晶体薄膜施加朝向基底压力的点，反射光谱蓝移；在所述待测细胞施加背向基底拉力的点，反射光谱红移，从而在成像组件中形成图案；
    所述成像组件，用于接收来自所述光子晶体基底的反射光进行成像，以利用成像图形得到所述待测细胞与所述光子晶体基底之间的作用力信息；
    所述成像组件包括光谱仪和相机，具体用于：
    使用纳米精度的压台对所述光子晶体薄膜进行挤压，使用所述光谱仪和所述相机同时记录光谱和彩色照片数据，再将同一时刻光谱的峰值位置和照片的色相进行对应，得到光子晶体反射峰位置和色相的映射关系；
    根据相差模式获得常规的相差照片，使用反射模式获得力学模态图，提取色相信息后根据色相与光子禁带峰位置的映射关系得到应变信息。
  2. 根据权利要求1所述的光子晶体显微镜，其特征在于，所述光子晶体薄膜在430nm～700nm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70％。
  3. 根据权利要求1所述的光子晶体显微镜，其特征在于，所述光子晶体薄膜的厚度范围为20μm～80μm。
  4. 根据权利要求1所述的光子晶体显微镜，其特征在于，所述光子晶体薄膜的杨氏模量范围为0.5kPa～100kPa。
  5. 根据权利要求1所述的光子晶体显微镜，其特征在于，所述光子晶体薄膜的光子禁带范围为430nm～700nm。
  6. 根据权利要求1至5任一项所述的光子晶体显微镜，其特征在于，所述光子晶体显微镜还包括物镜，所述物镜设置在所述载物台和所述探测光源之间。
  7. 根据权利要求6所述的光子晶体显微镜，其特征在于，所述物镜的数值孔径范围为0.1～0.9，和/或，所述物镜的放大倍率范围为1倍～100倍。
  8. 根据权利要求1至5任一项所述的光子晶体显微镜，其特征在于，所述成像组件包括滤光器和感光元件，所述滤光器所在光路位于所述感光元件和所述载物台之间。
  9. 一种细胞力学测量方法，其特征在于：采用权利要求1至8任一项所述的光子晶体显微镜，所述方法包括：
    将所述光子晶体基底置于所述载物台上，观察所述光子晶体基底在待测细胞作用下发生形变；
    所述探测光源向所述光子晶体基底发出探测光；
    所述光子晶体基底反射所述探测光至所述成像组件；
    所述成像组件接收来自所述光子晶体基底的反射光进行成像，以利用成像图形得到所述待测细胞与所述光子晶体基底之间的作用力信息。
PCT/CN2020/137819 2020-09-08 2020-12-19 光子晶体显微镜和细胞力学测量方法 WO2022052361A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP20953146.6A EP4212857A1 (en) 2020-09-08 2020-12-19 Photonic crystal microscope and cell mechanics measurement method
US18/180,120 US20230221538A1 (en) 2020-09-08 2023-03-07 Photonic Crystal Microscope and Method of Measuring Cellular Forces

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010931803.4 2020-09-08
CN202010931803.4A CN111812095B (zh) 2020-09-08 2020-09-08 光子晶体显微镜和细胞力学测量方法

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/180,120 Continuation US20230221538A1 (en) 2020-09-08 2023-03-07 Photonic Crystal Microscope and Method of Measuring Cellular Forces

Publications (1)

Publication Number Publication Date
WO2022052361A1 true WO2022052361A1 (zh) 2022-03-17

Family

ID=72860029

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/137819 WO2022052361A1 (zh) 2020-09-08 2020-12-19 光子晶体显微镜和细胞力学测量方法

Country Status (4)

Country Link
US (1) US20230221538A1 (zh)
EP (1) EP4212857A1 (zh)
CN (1) CN111812095B (zh)
WO (1) WO2022052361A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111812095B (zh) * 2020-09-08 2020-12-25 东南大学苏州医疗器械研究院 光子晶体显微镜和细胞力学测量方法
CN115876759A (zh) * 2021-09-26 2023-03-31 瑞新（福州）科技有限公司 细胞机械力的检测系统、方法、装置及其制备方法
CN115039738A (zh) * 2022-04-07 2022-09-13 南京大学 一种光子晶体薄膜在减缓线虫光毒害中的应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533537A (zh) * 2011-12-29 2012-07-04 云南师范大学 一种高通量筛选细胞在三维基质中生长迁移行为的装置及方法
US20130230879A1 (en) * 2012-02-17 2013-09-05 Colorado School Of Mines Optical alignment deformation spectroscopy
CN105738254A (zh) * 2016-02-03 2016-07-06 苏州大学 一种力学生物学耦合测试系统及方法
CN109827928A (zh) * 2019-02-02 2019-05-31 东南大学 多模态生物力学显微镜及测量方法
CN111812095A (zh) * 2020-09-08 2020-10-23 东南大学苏州医疗器械研究院 光子晶体显微镜和细胞力学测量方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104359876B (zh) * 2014-10-14 2018-07-10 厦门大学 细胞牵引力显微镜及其在抗癌药物药效及药理检测中的应用
GB201421214D0 (en) * 2014-11-28 2015-01-14 Univ Aberdeen Micro-cavity-based force sensor
CN106404915B (zh) * 2016-08-29 2019-02-19 湖南农业大学 一种细胞牵引力的实时与定量测定方法
US11782046B2 (en) * 2017-07-03 2023-10-10 The Regents Of The University Of California Single-pixel optical technologies for instantly quantifying multicellular response profiles

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102533537A (zh) * 2011-12-29 2012-07-04 云南师范大学 一种高通量筛选细胞在三维基质中生长迁移行为的装置及方法
US20130230879A1 (en) * 2012-02-17 2013-09-05 Colorado School Of Mines Optical alignment deformation spectroscopy
CN105738254A (zh) * 2016-02-03 2016-07-06 苏州大学 一种力学生物学耦合测试系统及方法
CN109827928A (zh) * 2019-02-02 2019-05-31 东南大学 多模态生物力学显微镜及测量方法
CN111812095A (zh) * 2020-09-08 2020-10-23 东南大学苏州医疗器械研究院 光子晶体显微镜和细胞力学测量方法

Also Published As

Publication number Publication date
EP4212857A1 (en) 2023-07-19
US20230221538A1 (en) 2023-07-13
CN111812095B (zh) 2020-12-25
CN111812095A (zh) 2020-10-23

Similar Documents

Publication Publication Date Title
WO2022052361A1 (zh) 光子晶体显微镜和细胞力学测量方法
US20140295538A1 (en) Device and system for mechanical measurement of biomaterial
Grosberg et al. Ensembles of engineered cardiac tissues for physiological and pharmacological study: heart on a chip
US8599383B2 (en) Optical cytometry
WO2020155716A1 (zh) 多模态生物力学显微镜及测量方法
Huebsch et al. Fluorescent resonance energy transfer: a tool for probing molecular cell–biomaterial interactions in three dimensions
US20070086919A1 (en) Apparatus and method for detecting deformability of cells using spatially modulated optical force microscopy
US20090263850A1 (en) Method and system for measuring single cell mechanics using a modified scanning probe microscope
Li et al. Photo-responsive photonic hydrogel: in situ manipulation and monitoring of cell scaffold stiffness
Lidstone et al. Label-free imaging of cell attachment with photonic crystal enhanced microscopy
US10201054B2 (en) Optical detection device, its method for operating and computer program
Langer et al. Mechanical stimulation of individual stereocilia of living cochlear hair cells by atomic force microscopy
Reed et al. High throughput cell nanomechanics with mechanical imaging interferometry
Iyer et al. Towards nonspecific detection of malignant cervical cells with fluorescent silica beads
Roy et al. Microarray-facilitated mechanical characterization of breast tissue pathology samples using contact-mode atomic force microscopy (AFM)
JP5713251B2 (ja) 細胞判別方法、細胞判別用の参照データ生成方法、および細胞判別装置
Siamantouras et al. Examining cell-cell interactions in the kidney using AFM single-cell force spectroscopy
CN117074405A (zh) 细胞牵引力的测量方法
WO2023046166A1 (zh) 细胞机械力的检测系统、方法、装置及其制备方法
Notbohm Dynamics of cell–matrix mechanical interactions in three dimensions
Plant et al. Tools for quantitative and validated measurements of cells
Wang et al. The Study of Cell Motility by Cell Traction Force Microscopy (CTFM)
Stephan et al. Single-Cell Optical Distortion Correction and Label-Free 3D Cell Shape Reconstruction on Lattices of Nanostructures
Varol et al. Holographic Cell Stiffness Mapping Using Acoustic Stimulation
Reed et al. Applications of imaging interferometry

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20953146

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020953146

Country of ref document: EP

Effective date: 20230411