【作者】 王波；

【导师】 陈学文；

【作者基本信息】 华中科技大学 ， 光学， 2022， 硕士

【摘要】 随着对微观世界的研究更加深入,科学界对无标记的高灵敏度显微技术的需求日益增长。干涉散射显微术(Interferometric Scattering Microscopy,iSCAT)作为一种无需荧光标记的超灵敏光学显微技术,近年来取得了重要的成就。其利用样品散射光和共路参考光的干涉,能达到单分子的探测灵敏度,且不存在光闪烁、光漂白、光饱和等缺点,在纳米颗粒追踪、细胞膜动力学过程监测等应用场景中均有优异表现。iSCAT信号与散射体体积成正比,蛋白质分子比体积恒定,体积与质量基本成正比,故其iSCAT信号与分子质量成正比。基于此,iSCAT有望用于蛋白质分子的质量称量,本论文围绕使用iSCAT实现高精度的单个蛋白质分子质量称量开展了如下研究:1.将蛋白质分子体积和折射率的计算与iSCAT理论模型结合,能准确地评估单个蛋白质分子的iSCAT信号。可以有效地指导蛋白质分子质量称量实验。2.结合模型估算和实验测量,提出了准确确定蛋白质分子探测的最佳聚焦位置的方法:两步聚焦法。由于蛋白质的iSCAT信号远小于基底表面起伏导致的散斑背景,难以直接找到合适的聚焦位置探测蛋白质。因此本论文先用更易探测、信号更大的金纳米颗粒初步确定聚焦位置,再考虑蛋白质与金纳米颗粒的信号谷值位置的固有差异,从而确定蛋白质动态探测的最佳聚焦位置。3.利用包含聚焦反馈光路的iSCAT系统,通过有效的背景减除,成功探测到单个蛋白质分子。通过傅里叶空间滤波,提高探测信噪比,从而提高了蛋白质质量称量精度。使用不同平整度的基底对蛋白质进行称量,证明本论文的背景减除方法能消除基底表面起伏导致的散斑背景的影响。更多还原

【Abstract】 With the further study of the microscopic world,there is an increasing demand for high sensitivity and lable-free microscopy.As a kind of ultra-sensitive and label-free optical microscopy,interferometric scattering microscopy(iSCAT)has made many important achievements in recent years.Using the interference of scattering light of sample and reference light in common path,it can achieve the detection sensitivity of single molecule,and does not have the shortcomings of photoblinking,photobleaching,photosaturation and so on.It has excellent performance in nano-particle tracking,cell membrane dynamic process monitoring and other applications.The iSCAT signal is proportional to the scatterer’s volume and the specific volume of proteins is a constant,so the volume and mass can be regarded as proportional.Therefore,the iSCAT signal is proportional to the molecular mass and iSCAT is very suitable for mass weighing of single protein.In order to achieve high-accuracy mass weighing of single protein by iSCAT,the following studies are carried out in this thesis:1.The iSCAT signal of a single protein can be accurately evaluated by combining the calculation of volume and refractive index of single protein with the iSCAT theoretical model.It can guide mass weighing experiment of the single protein effectively.2.Combined estimation by simulated model with experimental measurement,a method is proposed to accurately determine the optimal focus position of detecting single protein.It is difficult to directly find the appropriate focus position because the iSCAT signal of single protein is much smaller than the speckle background caused by the fluctuation of the base surface.Through theoretical calculation and experimental measurement,the relationship between the optimal focus position for detection of the protein and gold nanoparticles can be obtained.Therefore,the optimal focus position for detection of single protein can be obtained with the auxiliary from gold nanoparticles that are much easier to be detected.3.Due to effectively background subtraction,the signal of single protein is successfully detected by the iSCAT,which contains focused feedback system.The signal-to-noise ratio(SNR)is improved and the accuracy of mass weighing of single protein is improved by Fourier space filtering.We also prove that the influence of speckle background caused by substrate surface roughness can be eliminated by the background subtraction.更多还原