CN111263086A - Method for manufacturing cell fusion photoelectric sensor and working method of imaging system thereof - Google Patents
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Abstract
本发明公开了一种细胞融合的光电传感器及其成像系统的工作方法。本发明细胞融合的光电传感器的制作方法,包括:将莱茵衣藻细胞眼点(Eye spot)处提取的视紫红质通道蛋白(ChR2)转染到人胚肾293(Hek293,Human embryonic kidney)细胞内;最后,通过荧光显微镜观测ChR2质粒在细胞中的表达,并用膜片钳技术在光刺激进一步验证细胞表达结果;以表达了ChR2的Hek293细胞为光电传感器的敏感材料,其将器件吸收的光信号转换成离子电流,并以膜片钳技术为该光电传感器的信号采集和处理单元;将该细胞与膜片钳检测设备相结合构成细胞融合光电传感器。本发明的有益效果:这种生物融合的光电器件能够充分利用生物体在光感知上或视觉上的优势,从而产生比纯人造装置性能更优新型器件。
The invention discloses a cell fusion photoelectric sensor and a working method of its imaging system. The manufacturing method of the cell-fused photoelectric sensor of the present invention includes: transfecting channelrhodopsin (ChR2) extracted from the eye spot of Chlamydomonas reinhardtii cells into human embryonic kidney 293 (Hek293, Human embryonic kidney) cells Finally, the expression of ChR2 plasmid in cells was observed by fluorescence microscope, and the results of cell expression were further verified by light stimulation with patch clamp technology; Hek293 cells expressing ChR2 were used as the sensitive material of the photoelectric sensor, which absorbed the light absorbed by the device. The signal is converted into ionic current, and the patch clamp technology is used as the signal acquisition and processing unit of the photoelectric sensor; the cell is combined with the patch clamp detection device to form a cell fusion photoelectric sensor. Beneficial effects of the present invention: The bio-integrated optoelectronic device can make full use of the organism's advantages in light perception or vision, thereby producing a new type of device with better performance than pure artificial devices.
Description
技术领域technical field
本发明涉及光电传感器领域,具体涉及一种细胞融合的光电传感器及其成像系统的工作方法。The invention relates to the field of photoelectric sensors, in particular to a cell fusion photoelectric sensor and a working method of an imaging system thereof.
背景技术Background technique
生物系统进过自然界亿万年的进化和发展有着许多人造系统所无法比拟的优势结构和功能特性。将生物细胞或组织与机电系统相融合构建生机融合系统能够充分利用生物体的结构和功能优势,从而创建出一些更加完善的结构和更加强大的功能特性。目前,在国际许多顶尖刊物上报道了许多以生物活体细胞或组织为驱动或能源的生机融合系统。加利福尼亚大学洛杉矶分校的Montemagno等人通过将肌细胞培养在微机械结构,装配成以肌肉驱动和控制的微型装置,实现微型装置的自由运动[J.Xi,J.J.Schmidt,C.D.Montemagno,Nat.Mater.2005,4,180.];哈佛大学的Parker等人将哺乳动物的心肌细胞装配在微机电系统(MEMS)上,以驱动微机械装置运动[A.W.Feinberg,A.Feigel,S.S.Shevkoplyas,S.Sheehy,G.M.Whitesides,K.K.Parker,Science 2007,317,1366.];伊利诺伊大学厄巴纳-香槟分校的Bashir等人用光遗传学技术修饰骨骼肌细胞,并通过光调节该细胞的伸缩特性以驱动3D打印的微机械装置的运动,该装置的直线移动距离可达到310um/s或每分钟1.3个身体长度的距离[R.Raman,C.Cvetkovic,S.G.Uzel,R.J.Platt,P.Sengupta,R.D.Kamm,R.Bashir,Proc.Natl.Acad.Sci.USA.2016,113,3497.]。此外,也有学者用嗅觉细胞和上皮组织与机电装置相融合构建生物传感器以提高传感器的灵敏度[L.Du,C.Wu,H.Peng,L.Zhao,L.Huang,P.Wang,Biosens.Bioelectron.2013,40,401.];用哺乳动物的味觉细胞和味觉蓓蕾构建的生物融合传感器能够检测到不同刺激物散发的多种味觉信号[Q.Liu,D.Zhang,F.Zhang,Y.Zhao,K.J.Hsia,P.Wang,Sens.Actuators,B2013,176,497.]。这些报道充分说明哺乳动物的活体细胞或组织能够作为非常有潜力的功能单元,用以构建生物驱动、生物嗅觉和味觉等。然而,目前还没有任何关于用活体细胞或组织构建生物融合的光电器件或光敏元件,尽管哺乳动物光敏细胞或组织具有许多人工光敏器件或视觉系统所无法比拟的优势,例如,视杆细胞具有感知单个光子的超高光响应度[D.A.Baylor,T.D.Lamb,K.W.Yau,J.Physiol.1979,288,613.],响尾蛇用1mm大小的颊窝实现红外感知捕获猎物[E.A.Newman,P.H.Hartline,Science 1981,213,789.]。利用动物视觉细胞或光敏细胞构建光敏器件能够有效提高器件的光敏特性。Biological systems have evolved and developed over hundreds of millions of years in nature, and have many superior structural and functional characteristics that are unmatched by man-made systems. Integrating biological cells or tissues with electromechanical systems to construct bio-integrated systems can make full use of the structural and functional advantages of organisms, thereby creating some more complete structures and more powerful functional properties. At present, many biological fusion systems using living cells or tissues as driving or energy sources have been reported in many top international journals. Montemagno et al. at the University of California, Los Angeles, achieved free movement of the microdevice by culturing muscle cells in a micromechanical structure and assembling it into a microdevice driven and controlled by muscles [J.Xi, J.J.Schmidt, C.D.Montemagno, Nat.Mater. 2005, 4, 180.]; Parker et al. of Harvard University assembled mammalian cardiomyocytes on a microelectromechanical system (MEMS) to drive the motion of the micromechanical device [A.W.Feinberg, A.Feigel, S.S.Shevkoplyas, S.Sheehy, G.M. Whitesides, K.K. Parker, Science 2007, 317, 1366.]; Bashir et al. of the University of Illinois at Urbana-Champaign modified skeletal muscle cells with optogenetics and modulated the cell's telescopic properties by light to drive 3D-printed Movement of a micromechanical device that can move linearly at a distance of 310 um/s or 1.3 body lengths per minute [R.Raman,C.Cvetkovic,S.G.Uzel,R.J.Platt,P.Sengupta,R.D.Kamm,R. Bashir, Proc. Natl. Acad. Sci. USA. 2016, 113, 3497.]. In addition, some scholars have used the fusion of olfactory cells and epithelial tissues with electromechanical devices to construct biosensors to improve the sensitivity of the sensors [L.Du,C.Wu,H.Peng,L.Zhao,L.Huang,P.Wang,Biosens. Bioelectron. 2013, 40, 401.]; A bio-fusion sensor constructed with mammalian taste cells and taste buds can detect a variety of taste signals emitted by different stimuli [Q.Liu, D. Zhang, F. Zhang, Y. Zhao, K. J. Hsia, P. Wang, Sens. Actuators, B2013, 176, 497.]. These reports fully demonstrate that mammalian living cells or tissues can be used as very potential functional units to construct biological drives, biological sense of smell and taste. However, there is currently nothing about constructing bio-fused optoelectronic devices or photosensors with living cells or tissues, although mammalian photosensitive cells or tissues have many advantages over artificial photosensors or visual systems. For example, rod cells have sensory Ultra-high photoresponsivity of a single photon [D.A.Baylor, T.D.Lamb, K.W.Yau, J.Physiol.1979,288,613.], Rattlesnake achieves infrared perception with 1mm-sized cheek socket to capture prey [E.A.Newman,P.H.Hartline,Science 1981,213,789 .]. Using animal visual cells or photosensitive cells to construct photosensitive devices can effectively improve the photosensitive properties of the devices.
此外,即使用光敏细胞构建了生物融合的光电器件,然而,要用该器件实现高分辨率成像依然存在一个不可跨越的问题。在传统的成像系统中,借助于CCD或CMOS等光敏元件阵列实现高分辨率成像,其中,CCD或CMOS的每个光敏元件对应获得的图像的一个像素点。然而,针对的这种生物融合的光电器件,目前的技术还难以制造出入CCD或CMOS一样的光电器件阵列,无发现传统成像系统一样实现高分辨率成像。In addition, even if photosensitive cells are used to construct bio-fused optoelectronic devices, however, there is still an insurmountable problem to achieve high-resolution imaging with this device. In traditional imaging systems, high-resolution imaging is achieved by means of photosensitive element arrays such as CCD or CMOS, wherein each photosensitive element of the CCD or CMOS corresponds to one pixel of the obtained image. However, for this kind of bio-integrated optoelectronic device, the current technology is still difficult to manufacture the same optoelectronic device array as CCD or CMOS, and it has not been found that the traditional imaging system can achieve high-resolution imaging.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题是提供一种细胞融合的光电传感器及其成像系统的工作方法,首次用活体细胞作为光敏单元构建了生物融合的光电传感器;针对目前技术难以实现新型光敏器件阵列化而无法实现高分辨率成像的问题,本发明构建了基于单个该生物融合光电器件的单像素成像系统,实现高分辨率的成像。The technical problem to be solved by the present invention is to provide a cell-fused photoelectric sensor and a working method of its imaging system. For the first time, a living cell is used as a photosensitive unit to construct a biologically fused photoelectric sensor; for the current technology is difficult to realize the array of new photosensitive devices and For the problem that high-resolution imaging cannot be achieved, the present invention constructs a single-pixel imaging system based on a single bio-fusion photoelectric device to achieve high-resolution imaging.
为了解决上述技术问题,本发明提供了一种细胞融合的光电传感器的制作方法,包括:将莱茵衣藻细胞眼点(Eye spot)处提取的视紫红质通道蛋白(ChR2)转染到人胚肾293(Hek293,Human embryonic kidney)细胞内;观测ChR2质粒在细胞中的表达,并用膜片钳技术在光刺激进一步验证细胞表达结果;以表达了ChR2的Hek293细胞为光电传感器的敏感材料,其将器件吸收的光信号转换成离子电流,并以膜片钳技术为该光电传感器的信号采集和处理单元;将该细胞与膜片钳检测设备相结合构成细胞融合光电传感器。In order to solve the above-mentioned technical problems, the present invention provides a method for manufacturing a photoelectric sensor of cell fusion, comprising: transfecting channelrhodopsin (ChR2) extracted from the eye spot of Chlamydomonas reinhardtii cells into human embryos Kidney 293 (Hek293, Human embryonic kidney) cells; observe the expression of ChR2 plasmid in cells, and use patch clamp technology to further verify the results of cell expression under light stimulation; Hek293 cells expressing ChR2 are used as sensitive materials for photoelectric sensors. The light signal absorbed by the device is converted into ionic current, and the patch clamp technology is used as the signal acquisition and processing unit of the photoelectric sensor; the cell is combined with the patch clamp detection device to form a cell fusion photoelectric sensor.
在其中一个实施例中,该人胚肾293(Hek293,Human embryonic kidney)细胞具体构建过程是:1)使用带有聚合酶(pfu,Promega)的校对聚合酶(带有BamHI和HindIII限制性酶切位点的引物),通过PCR从全长cDNA模板(GenBank号:AF461397)获得全长chop2-315和C末端截短的chop2突变,并将其制成ChR2质粒;2)将Hek293细胞培养在9.6cm2的培养皿中,其中含有10%胎牛血清和1%双抗的高糖DMEM培养基,并将培养皿放置在37℃、含5%浓度的CO2的培养箱中,待细胞长满培养皿的1/2时备用;3)利用转染试剂(Lipofectamine2000,Sigma)将ChR2质粒转染到Hek293细胞中,并放置在细胞培养箱中培养24小时;4)在培养皿中加入1uM的顺式视黄醛(all-trans retinal,Sigma),并在培养箱中孵育2小时。In one embodiment, the specific construction process of the human embryonic kidney 293 (Hek293, Human embryonic kidney) cells is: 1) using a proofreading polymerase (with BamHI and HindIII restriction enzymes) with a polymerase (pfu, Promega) The primers for the cut site), the full-length chop2-315 and the C-terminal truncated chop2 mutation were obtained from the full-length cDNA template (GenBank No.: AF461397) by PCR and made into a ChR2 plasmid; 2) Hek293 cells were cultured in In a 9.6cm2 petri dish containing high glucose DMEM medium containing 10% fetal bovine serum and 1% double antibody, the petri dish was placed in an incubator at 37°C with 5% CO2 , and the cells were 3) Use transfection reagent (Lipofectamine2000, Sigma) to transfect the ChR2 plasmid into Hek293 cells, and place them in a cell incubator for 24 hours; 4) Add to the
在其中一个实施例中,通过荧光显微镜观测ChR2质粒在细胞中的表达。In one embodiment, the expression of the ChR2 plasmid in the cells is observed by fluorescence microscopy.
一种基于细胞融合的光电传感器的成像系统的工作方法,光透过目标图像后,经过第一透镜汇聚到光空间调制器DMD上,通过随机二值图像控制DMD中相应的每一个微镜的翻转以控制反射到第二透镜上面的光强,并汇聚于任一项制作方法制作的生物融合的光电传感器,同时用膜片钳检测细胞的光响应信号;最后,利用膜片钳检测的信号重构出原始图像。A working method of an imaging system based on a photoelectric sensor based on cell fusion. After the light passes through the target image, it is concentrated on the optical spatial modulator DMD through a first lens, and the corresponding micromirror in the DMD is controlled by a random binary image. Flip to control the light intensity reflected on the second lens, and focus on the bio-fused photoelectric sensor fabricated by any of the fabrication methods, and use patch clamp to detect the light response signal of the cell; finally, use the patch clamp to detect the signal The original image is reconstructed.
在其中一个实施例中,其中,成像系统的成像方法是基于压缩感知原理,以单个光电探测器实现高分辨率的成像;压缩感知原理可描述为:对于某未知信号x∈RN×1,在测量矩阵Φ∈RM×N(M<N)下的线性观测值为y∈RM×1,则有:In one of the embodiments, the imaging method of the imaging system is based on the principle of compressed sensing, and a single photodetector realizes high-resolution imaging; the principle of compressed sensing can be described as: for an unknown signal x∈R N×1 , The linear observation value under the measurement matrix Φ∈R M×N (M<N) is y∈R M×1 , then:
y=Φx; (1)y=Φx; (1)
其中,信号x必需满足稀疏性条件,即x或x转换域内只有K(K<M)个元素为非零值;根据信号x的稀疏性,从满足式(1)的解中找出最稀疏的,当测量矩阵Φ满足限制性等距性质(Restricted Isometry Property,RIP:(1-δ)||x||2 2≤||Φx||2 2≤(1+δ)||x||2 2)时,这个“最稀疏”的解就是原始信号x;在该系统中,基于压缩感知原理通过DMD将图像的空间信号转换成时间系列信号,然后获得重构图像。Among them, the signal x must meet the sparsity condition, that is, only K (K<M) elements in the transformation domain of x or x are non-zero values; according to the sparsity of the signal x, find the most sparse solution from the solution satisfying Eq. , when the measurement matrix Φ satisfies the Restricted Isometric Property (RIP: (1-δ)||x|| 2 2 ≤||Φx|| 2 2 ≤(1+δ)||x|| 2 2 ), the "sparsest" solution is the original signal x; in this system, based on the principle of compressed sensing, the spatial signal of the image is converted into a time series signal through DMD, and then the reconstructed image is obtained.
在其中一个实施例中,压缩感知原理具体的过程是:1)假设要获得的信号x,长度为N,测量矩阵Φ∈RM×N(M<N)设计为伯努利随机矩阵,其中每位元素都为1或0;2)将测量矩阵的每一行映射成一个随机二值图像,该图像大小与目标图像尺寸一致;3)依次用随机二值图像控制DMD的翻转,并通过膜片钳检测每次翻转后细胞的离子电流信号,其信号大小可表示为其中,j=1,2,...,M,Ii(i=1,2,3,...,N)在该微镜偏向二极管方向是为1,反之为0,而Dcoffset是所有微镜都偏离石墨烯器件方向的测量值;最后,通过从获得的采样信号vi和测量矩阵Φ∈RM×N重构出原始图像x。In one of the embodiments, the specific process of the compressed sensing principle is: 1) Assuming that the signal x to be obtained has a length of N, the measurement matrix Φ∈R M×N (M<N) is designed as a Bernoulli random matrix, where Each element is 1 or 0; 2) Map each row of the measurement matrix into a random binary image whose size is consistent with the size of the target image; 3) Control the inversion of the DMD with the random binary image in turn, and pass the film Patch clamp detects the ion current signal of cells after each flip, and the signal size can be expressed as Among them, j=1,2,...,M, I i (i=1,2,3,...,N) is 1 when the micromirror is deflected to the diode direction, otherwise it is 0, and Dcoffset is all The micromirrors are all deviated from the measured values of the graphene device orientation; finally, the original image x is reconstructed from the obtained sampled signal vi and the measurement matrix Φ∈R M×N .
在其中一个实施例中,通过压缩感知重构算法从获得的采样信号vi和测量矩阵Φ∈RM×N重构出原始图像x。In one of the embodiments, the original image x is reconstructed from the obtained sampled signal v i and the measurement matrix Φ∈R M×N by a compressive sensing reconstruction algorithm.
基于同样的发明构思,本申请还提供一种计算机设备,包括存储器、处理器及存储在存储器上并可在处理器上运行的计算机程序,所述处理器执行所述程序时实现任一项所述方法的步骤。Based on the same inventive concept, the present application also provides a computer device, including a memory, a processor, and a computer program stored in the memory and running on the processor, the processor implements any one of the above when executing the program. steps of the method described.
基于同样的发明构思,本申请还提供一种计算机可读存储介质,其上存储有计算机程序,该程序被处理器执行时实现任一项所述方法的步骤。Based on the same inventive concept, the present application also provides a computer-readable storage medium on which a computer program is stored, and when the program is executed by a processor, implements the steps of any one of the methods.
基于同样的发明构思,本申请还提供一种处理器,所述处理器用于运行程序,其中,所述程序运行时执行任一项所述的方法。Based on the same inventive concept, the present application also provides a processor for running a program, wherein the program executes any one of the methods when the program runs.
本发明的有益效果:Beneficial effects of the present invention:
该传感器用光敏的活体细胞作为感光元件,这种生物融合的光电器件能够充分利用生物体在光感知上或视觉上的优势,从而产生比纯人造装置性能更优新型器件,例如,该细胞融合的光电传感器具有良好的光适应性和更高的饱和度;此外,构建的成像系统首次用细胞融合的光电传感器实现了对宏观物体的高分辨率成像。The sensor uses photosensitive living cells as photosensitive elements. This bio-fused optoelectronic device can make full use of the organism's advantages in light perception or vision, resulting in new devices with better performance than pure artificial devices. For example, the cell fusion The photosensors have good light adaptability and higher saturation; in addition, the constructed imaging system realizes high-resolution imaging of macroscopic objects for the first time with cell-fused photosensors.
附图说明Description of drawings
图1是本发明细胞融合的光电传感器构建的原理示意图。FIG. 1 is a schematic diagram of the construction of the photoelectric sensor for cell fusion of the present invention.
图2是本发明细胞融合的光电传感器的成像系统的结构简图。FIG. 2 is a schematic structural diagram of the imaging system of the photoelectric sensor for cell fusion of the present invention.
图3是本发明中的成像结果示意图。其中,A~E、目标图像;F~J、细胞融合光电探测器成像结果。FIG. 3 is a schematic diagram of the imaging result in the present invention. Among them, A~E, target image; F~J, cell fusion photodetector imaging result.
图4是本发明中的细胞融合光电探测器成像结果与一个商业化光电探测器成像结果对比。FIG. 4 is a comparison between the imaging results of the cell fusion photodetector in the present invention and the imaging results of a commercial photodetector.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
本发明提供了一种细胞融合的光电传感器及其成像系统。细胞融合的光电传感器构建过程如图1所示。将莱茵衣藻细胞眼点(Eye spot)处提取的视紫红质通道蛋白(ChR2)转染到人胚肾293(Hek293,Human embryonic kidney)细胞内,该细胞具有良好的光敏特性,并且通过膜片钳技术能够检测出细胞的光响应特性。该光敏细胞具体构建过程是:1)使用带有聚合酶(pfu,Promega)的校对聚合酶(带有BamHI和HindIII限制性酶切位点的引物),通过PCR从全长cDNA模板(GenBank号:AF461397)获得全长chop2-315和C末端截短的chop2突变,并将其制成ChR2质粒;2)将Hek293细胞培养在9.6cm2的培养皿中,其中含有10%胎牛血清和1%双抗的高糖DMEM培养基,并将培养皿放置在37℃、含5%浓度的CO2的培养箱中,待细胞长满培养皿的1/2时备用;3)利用转染试剂(Lipofectamine 2000,Sigma)将ChR2质粒转染到Hek293细胞中,并放置在细胞培养箱中培养24小时;4)在培养皿中加入1uM的顺式视黄醛(all-trans retinal,Sigma),并在培养箱中孵育2小时。最后,通过荧光显微镜观测ChR2质粒在细胞中的表达,并用膜片钳技术在光刺激进一步验证细胞表达结果。以表达了ChR2的Hek293细胞为光电传感器的敏感材料,其将器件吸收的光信号转换成离子电流,并以膜片钳技术为光电传感器的信号采集和处理单元。将该细胞与膜片钳检测设备相结合构成细胞融合的光电传感器。The invention provides a photoelectric sensor for cell fusion and an imaging system thereof. The photoelectric sensor construction process for cell fusion is shown in Figure 1. The channelrhodopsin protein (ChR2) extracted from the eye spot of Chlamydomonas reinhardtii cells was transfected into human embryonic kidney 293 (Hek293, Human embryonic kidney) cells, which have good photosensitivity and can pass through the membrane. The patch-clamp technique can detect the light-responsive properties of cells. The specific construction process of the photosensitive cell is: 1) Using a proofreading polymerase with a polymerase (pfu, Promega) (primers with BamHI and HindIII restriction sites), a full-length cDNA template (GenBank No. : AF461397) to obtain full-length chop2-315 and C-terminal truncated chop2 mutation and make it into a ChR2 plasmid; 2 ) to culture Hek293 cells in a 9.6 cm dish containing 10% fetal bovine serum and 1 % double-antibody high-glucose DMEM medium, and place the culture dish in an incubator at 37°C with 5% CO 2 until the cells reach 1/2 of the culture dish; 3) Use transfection reagent (Lipofectamine 2000, Sigma) The ChR2 plasmid was transfected into Hek293 cells and placed in a cell incubator for 24 hours; 4) 1uM cis-retinal (all-trans retinal, Sigma) was added to the culture dish, and incubated in the incubator for 2 hours. Finally, the expression of ChR2 plasmid in cells was observed by fluorescence microscope, and the results of cell expression were further verified by light stimulation with patch clamp technique. Hek293 cells expressing ChR2 are used as the sensitive material of the photoelectric sensor, which converts the light signal absorbed by the device into ionic current, and the patch clamp technology is used as the signal acquisition and processing unit of the photoelectric sensor. The cells were combined with a patch-clamp detection device to form a photoelectric sensor for cell fusion.
基于该细胞融合光电探测器的单像素成像系统结构如图2所示。光透过目标图像后,经过第一透镜汇聚到光空间调制器DMD上,通过随机二值图像控制DMD中相应的每一个微镜的翻转以控制反射到第二透镜上面的光强,并汇聚于生物融合的光电传感器,同时用膜片钳检测细胞的光响应信号。最后,膜片钳检测的信号通过优化算法重构出原始图像。The structure of the single-pixel imaging system based on the cell fusion photodetector is shown in Figure 2. After the light passes through the target image, it converges on the optical spatial modulator DMD through the first lens, and controls the flip of each corresponding micromirror in the DMD through the random binary image to control the light intensity reflected on the second lens, and converges Photoelectric sensors for biofusion, while using patch clamp to detect the light response signal of cells. Finally, the signal detected by the patch clamp is reconstructed from the original image by an optimized algorithm.
成像方法是基于压缩感知原理,以单个光电探测器实现高分辨率的成像。压缩感知原理可描述为:对于某未知信号x∈RN×1,在测量矩阵Φ∈RM×N(M<N)下的线性观测值为y∈RM×1,则有:The imaging method is based on the principle of compressed sensing to achieve high-resolution imaging with a single photodetector. The principle of compressed sensing can be described as: for an unknown signal x∈R N×1 , the linear observation value under the measurement matrix Φ∈R M×N (M<N) is y∈R M×1 , then:
y=Φx。 (1)y=Φx. (1)
其中,信号x必需满足稀疏性条件,即x或x转换域内只有K(K<M)个元素为非零值。由线性代数理论可知,方程(1)有无穷多个解,无法从观测值y中唯一确定原始信号x。然而,根据信号x的稀疏性,从满足式(1)的解中找出最稀疏的,当测量矩阵Φ满足限制性等距性质(Restricted Isometry Property,RIP:(1-δ)||x||2 2≤||Φx||2 2≤(1+δ)||x||2 2)时,这个“最稀疏”的解就是原始信号x。在该系统中,基于压缩感知原理通过DMD将图像的空间信号转换成时间系列信号,然后,通过优化算法获得重构图像,该图像能够以极高概率与原始图像信息相一致。具体的过程是:1)假设要获得的信号x,长度为N,测量矩阵Φ∈RM×N(M<N)设计为伯努利随机矩阵,其中每位元素都为1或0;2)将测量矩阵的每一行映射成一个随机二值图像,该图像大小与目标图像尺寸一致;3)依次用随机二值图像控制DMD的翻转,并通过膜片钳检测每次翻转后细胞的离子电流信号,其信号大小可表示为其中,j=1,2,...,M,Ii(i=1,2,3,...,N)在该微镜偏向二极管方向是为1,反之为0,而Dcoffset是所有微镜都偏离石墨烯器件方向的测量值;最后,通过压缩感知重构算法从获得的采样信号vi和测量矩阵Φ∈RM×N重构出原始图像x。Among them, the signal x must satisfy the sparsity condition, that is, only K (K<M) elements in the transformation domain of x or x are non-zero values. According to linear algebra theory, equation (1) has infinitely many solutions, and the original signal x cannot be uniquely determined from the observed value y. However, according to the sparsity of the signal x, find the most sparse solution from the solutions satisfying Eq. (1), when the measurement matrix Φ satisfies the Restricted Isometric Property (RIP: (1-δ)||x| | 2 2 ≤||Φx|| 2 2 ≤(1+δ)||x|| 2 2 ), the “sparsest” solution is the original signal x. In this system, based on the principle of compressed sensing, the spatial signal of the image is converted into a time series signal by DMD, and then the reconstructed image is obtained through the optimization algorithm, and the image can be consistent with the original image information with a very high probability. The specific process is: 1) Assuming that the signal x to be obtained is of length N, the measurement matrix Φ∈R M×N (M<N) is designed as a Bernoulli random matrix, in which each element is 1 or 0; 2 ) Map each row of the measurement matrix into a random binary image whose size is consistent with the size of the target image; 3) Control the inversion of the DMD with the random binary image in turn, and detect the ions of the cells after each inversion by patch clamp The current signal, its signal size can be expressed as Among them, j=1,2,...,M, I i (i=1,2,3,...,N) is 1 when the micromirror is deflected to the diode direction, otherwise it is 0, and Dcoffset is all The micromirrors are all deviated from the measured value of the graphene device direction; finally, the original image x is reconstructed from the obtained sampled signal vi and the measurement matrix Φ∈R M×N by the compressive sensing reconstruction algorithm.
下面介绍一个本发明的具体应用场景:A specific application scenario of the present invention is introduced below:
制造细胞融合的光电探测器的前提在于构建光敏细胞。用转染试剂(Lipofectamine 2000)将ChR2质粒转染到Hek293细胞,其中,ChR2质粒与培养基(高糖DMEM)比例是8ug:0.5mL,Lipofectamine与培养基的比例是20uL:0.5mL。将转染后的Hek293细胞放置在37℃、含5%浓度的CO2的培养箱中孵育24小时候,按顺式视黄醛与培养基为1uL:1mL的比例加入顺式视黄醛,孵育2小时候用于构建细胞融合生物传感器。The premise of making photodetectors for cell fusion lies in the construction of light-sensitive cells. The ChR2 plasmid was transfected into Hek293 cells with transfection reagent (Lipofectamine 2000), wherein the ratio of ChR2 plasmid to medium (high glucose DMEM) was 8ug:0.5mL, and the ratio of Lipofectamine to medium was 20uL:0.5mL. The transfected Hek293 cells were placed at 37°C in an incubator with a concentration of 5% CO2 for 24 hours, and cis-retinal was added according to the ratio of cis-retinal and medium to 1uL: 1mL, and incubated for 2 hours. Used to build cell fusion biosensors as a child.
用膜片钳装置解析光敏细胞的光响应特性,需要保证细胞内外渗透压并维持细胞长时间的活性,因此,膜片钳的玻璃管内外溶液需要用模拟生理环境的液体。胞外溶液的具体的配置是:140mM的NaCl、1mM的CaCl2、2mM的MgCl2和10mM的HEPES,并用NaOH将溶液的pH值调制7.4(室温);胞内溶液的具体的配置是:140mM的NaCl、5mM的EGTA、2mM的MgCl2和10mM的HEPES,并用NaOH将溶液的pH值调制7.4(室温)。此外,在检测该细胞光电流之前,先将细胞消化后制成悬浮液,并用多聚赖氨酸将细胞固定在载玻片上,然后,在细胞培养箱中孵育2小时左右。最后,将载玻片放置在移至装有胞外溶液的培养皿中,并放置在膜片钳操作台上。调节成像系统入射光,并检测该光敏细胞的光响应特性。用膜片钳检测细胞离子电流模式是在电压钳下记录全细胞电流,玻璃管是硼硅玻璃毛细管,内径0.5mm外径1mm,经玻璃管拉制造后,玻璃管入液电阻为3.5~5MΩ。采样频率为10kHz,并用2kHz低通滤波处理采样数据。封接电阻必须高于1GΩ,同时补偿液位电阻和电容,以保证细胞离子电流采样准确性和精度。To analyze the light response characteristics of photosensitive cells with a patch clamp device, it is necessary to ensure the osmotic pressure inside and outside the cells and maintain the cell activity for a long time. Therefore, the solution inside and outside the glass tube of the patch clamp needs to use a liquid that simulates the physiological environment. The specific configuration of the extracellular solution is: 140 mM NaCl, 1 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES, and the pH of the solution is adjusted to 7.4 (room temperature) with NaOH; the specific configuration of the intracellular solution is: 140 mM NaCl , 5mM EGTA, 2mM MgCl2 and 10mM HEPES, and adjusted the pH of the solution to 7.4 (room temperature) with NaOH. In addition, before measuring the cell photocurrent, the cells were digested and made into a suspension, and the cells were fixed on a glass slide with polylysine, and then incubated in a cell incubator for about 2 hours. Finally, place the slide in a petri dish containing the extracellular solution and place it on the patch clamp stage. The incident light of the imaging system is adjusted, and the photoresponse characteristics of the photosensitive cells are detected. Using patch clamp to detect cell ion current mode is to record whole cell current under voltage clamp, glass tube is borosilicate glass capillary, inner diameter is 0.5mm and outer diameter is 1mm. . The sampling frequency is 10kHz, and the sampled data is processed with 2kHz low-pass filtering. The sealing resistance must be higher than 1GΩ, while compensating for liquid level resistance and capacitance to ensure the accuracy and precision of cell ion current sampling.
DMD模块直接使用DLP芯片的Discovery系列。DMD类型为0.7英寸VGA系列,包含1024×768个数字微镜,每个微镜边长为13.7μm,适用于紫外到近红外的所有波段。控制板刷新率最高可达290Hz。每个微镜都能沿着微镜对角线向两边翻转12°,并通过输入的响应数值控制。当该位置的输入为1时,微镜向一个方向翻转12°;当该位置的输入为0时,微镜向另一个方向翻转12°。整个DMD的所有微镜能够用一张1024×768像素的随机二值图片同时控制,图片的像素点与DMD的微镜一一对应。The DMD module directly uses the Discovery series of DLP chips. The DMD type is a 0.7-inch VGA series, including 1024×768 digital micromirrors, each with a side length of 13.7μm, suitable for all wavelengths from ultraviolet to near-infrared. The control board refresh rate can be up to 290Hz. Each micromirror can be flipped 12° to both sides along the diagonal of the micromirror, and is controlled by the input response value. When the input at this position is 1, the micromirror is flipped 12° in one direction; when the input at this position is 0, the micromirror is flipped 12° in the other direction. All the micromirrors of the entire DMD can be controlled simultaneously with a random binary image of 1024×768 pixels, and the pixels of the image correspond to the micromirrors of the DMD one-to-one.
光源采用的是波长为375nm的激光器,以保证光线的直线性。并用10倍扩束器增加出瞳直径,以保证光线能够覆盖住DMD。The light source uses a laser with a wavelength of 375 nm to ensure the linearity of the light. And use a 10x beam expander to increase the diameter of the exit pupil to ensure that the light can cover the DMD.
在成像过程中以恒定的光强照射到DMD上,依次用相应的随机二值图片控制着DMD以100ms的周期的速度翻转,其中,DMD按相应图片翻转的停留时间为10ms。针对这个实施例中的光敏细胞,在10ms光脉冲刺激下,细胞离子电流从被激活到失活的整个过程时长约为77ms。为了保证每次检测到的光响应信号的稳定性和准确性,DMD翻转周期应大于77ms。During the imaging process, the DMD is irradiated with a constant light intensity, and the corresponding random binary images are used to control the DMD to flip at a speed of 100ms period, wherein the DMD is flipped according to the corresponding image for a dwell time of 10ms. For the photosensitive cells in this example, under the stimulation of 10ms light pulses, the entire process duration of the cell ion current from being activated to inactivating is about 77ms. In order to ensure the stability and accuracy of the optical response signal detected each time, the DMD turnover period should be greater than 77ms.
以图3A~E的字符图形作为目标图像,依次对其进行成像测试。成像结果如图3F~J所示。重构图形分辨率为50×50,采样率为30%。测量矩阵设计为750×2500的伯努利矩阵,其中,每个元素随机设置为1或者0。测量矩阵的每一行转换成一个50×50的模式矩阵(),然后,将其按比例扩增成1000×700matr i x()控制DMD中心区域,而DMD周边的剩余微镜对应的数据以0补充,最后,生成相应的1024×768的随机二值图片。而后将膜片钳检测的数据和测量矩阵,通过正交匹配追踪算法生成重构图像(图3F~J)。Taking the character patterns in Figs. 3A to E as target images, imaging tests were performed on them in turn. The imaging results are shown in Figures 3F–J. The reconstructed graphics have a resolution of 50×50 and a sampling rate of 30%. The measurement matrix is designed as a 750×2500 Bernoulli matrix, in which each element is randomly set to 1 or 0. Each row of the measurement matrix is converted into a 50 × 50 pattern matrix ( ), which is then scaled up to 1000 × 700 matr i x ( ) to control the central area of the DMD, and the data corresponding to the remaining micromirrors around the DMD are supplemented with 0 , and finally, generate the corresponding 1024×768 random binary image. Then, the patch-clamped data and measurement matrix are used to generate reconstructed images through the orthogonal matching pursuit algorithm (Fig. 3F-J).
为了更直观地观测细胞融合光电探测器的成像效果,以一个商业化的光电二极管作为成像系统的感光元件,对字符“I”的图形进行成像,结果如图4所示。在该实施例中,光源功率分别设置为0.8mW和8mW的,并用着两种探测器在相同条件下分别成像。从结果图4可知,在低的光功率下,两种光电器件都能够清晰地复现出目标图像的信息;而在高的光功率(8mW)下,光电二极管已经无法成像,而细胞融合的光电探测器依然能够较清晰地复现目标形状。在该实施例中,初步凸显了生物融合传感器相对人造器件的优势。In order to observe the imaging effect of the cell fusion photodetector more intuitively, a commercial photodiode was used as the photosensitive element of the imaging system to image the pattern of the character "I". The results are shown in Figure 4. In this example, the light source powers were set to 0.8 mW and 8 mW, respectively, and two kinds of detectors were used to image separately under the same conditions. From the results in Figure 4, it can be seen that at low optical power, both optoelectronic devices can clearly reproduce the information of the target image; while at high optical power (8mW), the photodiode can no longer image, and the cell fusion The photodetector can still reproduce the target shape relatively clearly. In this embodiment, the advantages of bio-fusion sensors over artificial devices are preliminarily highlighted.
以上对本发明提供的细胞融合的光电传感器及其成像系统的工作方法做了详细的描述,还有以下几点需要说明:The working method of the photoelectric sensor for cell fusion and its imaging system provided by the present invention has been described in detail above, and the following points need to be explained:
本发明提供了一种基于生物融合光电传感器的单像素成像系统,其特征包括:光源、目标图像、第一透镜、DMD、第二透镜、细胞融合光电传感器和电脑。光源发出的光投射过目标图像后,经过第一透镜汇聚在DMD上,再经DMD反射后经过第二透镜汇聚于光电传感器的光敏细胞上,光敏细胞的光电信号被膜片钳采集后传输到电脑,通过优化算法复现出重构图像。The invention provides a single-pixel imaging system based on a bio-fusion photoelectric sensor, which is characterized by comprising: a light source, a target image, a first lens, a DMD, a second lens, a cell fusion photoelectric sensor and a computer. After the light emitted by the light source is projected through the target image, it is concentrated on the DMD through the first lens, and then reflected by the DMD and then concentrated on the photosensitive cells of the photoelectric sensor through the second lens. The photoelectric signals of the photosensitive cells are collected by the patch clamp and transmitted to The computer reproduces the reconstructed image through an optimized algorithm.
细胞融合光电传感器包括光敏细胞和膜片钳工具。光敏细胞是用光遗传学工具将视紫红质通道蛋白(ChR2)修饰的人胚胎细胞(Hek293)构成。Cell fusion photosensors include photosensitive cells and patch clamp tools. The photosensitive cells are composed of human embryonic cells (Hek293) modified with channelrhodopsin (ChR2) using optogenetic tools.
膜片钳的玻璃管是硼硅玻璃毛细管,内径0.5mm外径1mm,经玻璃管拉制造后,玻璃管入液电阻为3.5~5MΩ。光敏细胞的光电流采用全细胞记录模式以提高细胞的响应幅值。The glass tube of the patch clamp is a borosilicate glass capillary with an inner diameter of 0.5 mm and an outer diameter of 1 mm. After the glass tube is drawn and manufactured, the resistance of the glass tube into liquid is 3.5-5MΩ. The photocurrent of photosensitive cells was recorded in whole-cell mode to increase the cell's response amplitude.
单像素成像基于压缩感知原理,具体的成像过程为:1)假设要获得的信号x,长度为N,测量矩阵Φ∈RM×N(M<N)设计为伯努利随机矩阵,其中每位元素都为1或0;2)将测量矩阵的每一行映射成一个随机二值图像,该图像大小与目标图像尺寸一致;3)依次用随机二值图像控制DMD的翻转,并通过膜片钳检测每次翻转后细胞的离子电流信号,其信号大小可表示为其中,j=1,2,...,M,Ii(i=1,2,3,...,N)在该微镜偏向二极管方向是为1,反之为0,而Dcoffset是所有微镜都偏离石墨烯器件方向的测量值;最后,通过压缩感知重构算法从获得的采样信号vi和测量矩阵Φ∈RM×N重构出原始图像x。Single-pixel imaging is based on the principle of compressed sensing. The specific imaging process is as follows: 1) Assuming that the signal x to be obtained is of length N, the measurement matrix Φ∈R M×N (M<N) is designed as a Bernoulli random matrix, in which each The bit elements are all 1 or 0; 2) Map each row of the measurement matrix into a random binary image whose size is consistent with the size of the target image; 3) Use the random binary image to control the inversion of the DMD in turn, and pass the diaphragm The clamp detects the ionic current signal of the cells after each flip, and the signal size can be expressed as Among them, j=1,2,...,M, I i (i=1,2,3,...,N) is 1 when the micromirror is deflected to the diode direction, otherwise it is 0, and Dcoffset is all The micromirrors are all deviated from the measured value of the graphene device direction; finally, the original image x is reconstructed from the obtained sampled signal vi and the measurement matrix Φ∈R M×N by the compressive sensing reconstruction algorithm.
电脑用于生成测量矩阵、随机二值图像、膜片钳控制软件,DMD控制软件、存储和处理膜片钳采样数据和重构图像。The computer is used to generate measurement matrices, random binary images, patch clamp control software, DMD control software, store and process patch clamp sampling data and reconstruct images.
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention is subject to the claims.
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