【作者】 刘俊；

【导师】 曾绍群； 黄振立；

【作者基本信息】 华中科技大学 ， 生物医学工程， 2011， 博士

【摘要】 光学显微成像是生命科学领域最主要的研究手段。但由于光的衍射现象,光学显微镜不能分辨尺寸小于200 nm的生物结构。近年出现了称为超分辨显微成像的技术,能实现分辨率达20 nm的光学成像。其中的一种广受关注的技术为光激活定位成像(Photoacitvated localization microscopy),这种成像技术主要包括新的光激活与成像光路,高灵敏度的相机,重建算法以及一类新产生的荧光标记物——高亮荧光蛋白(Optical highlighter fluorescent proteins)。该类蛋白被特定波长的光激活后,吸收和发射光谱特性会发生显著改变。合理的光激活定位成像技术依赖于光激活性能优异的高亮荧光蛋白,同时高亮荧光蛋白本身的光激活特性也是实际光激活定位成像实验中挑选合适的高亮荧光蛋白以及合理控制激活条件的依据。然而,由于光激活过程受到激活光强的影响明显,导致不同实验条件下进行的光激活实验结果相互间不可比较,因此目前并没有一个合理的用于表征高亮荧光蛋白光激活特性的参数,也没有用于实际实验时挑选及控制蛋白的指导。本文基于荧光蛋白的光漂白特性表征方法,提出了光强依赖的光激活与光漂白速率参数,用以表征高亮荧光蛋白的激活特性。并用此参数表征了四种典型的高亮荧光蛋白,并对四种蛋白的光强依赖的光激活速率差异进行了激活机理上的解释。进一步,通过将光动力治疗中的光剂量概念引入原有激活荧光表达式,得到了高亮荧光蛋白的光剂量荧光曲线表达式,并依此给出了所研究的四种高亮荧光蛋白的光剂量荧光曲线。最后,针对现有光激活定位成像方法,本文利用表征高亮蛋白不同激活光强下的光激活特性优化了现有的激活控制方法,改进后的方法可以将激活过程总时间减少50%,同时不影响成像质量。本文取得的主要研究结果如下:(1)提出了光强依赖的光激活与光漂白速率参数,用以表征高亮荧光的光激活特性。通过分析高亮荧光蛋白在不同光强激活光作用下光激活和光漂白过程,基于荧光蛋白光漂白特性研究及其中对荧光蛋白光漂白特性表征方法,考虑了实际光激活成像实验中对荧光蛋白光漂白特性影响最主要的条件—激发光强对高亮荧光蛋白激活特性的影响,提出了光强依赖的激活与漂白速率这一新表征参数,此参数能对高亮荧光蛋白的光激活特性进行更接近其物理本质的表征。用此参数表征几种典型高亮荧光蛋白的光激活特性的结果说明,根据激活机理的不同,不同蛋白的光强依赖的光激活速率参数存在明显差异。(2)提出了光剂量荧光曲线,用以直观、定量评价高亮荧光蛋白的光激活性能,可以用于比较不同高亮荧光蛋白的激活对比度及在激活光作用下荧光变化的快慢,为实际的光激活成像实验提供指导。本文分析并展示了将光剂量概念引入高亮荧光蛋白激活过程的调控研究。通过实验结果和理论推导,得到了高亮荧光蛋白荧光强度与激活光剂量的关系式,以及几种典型高亮荧光蛋白的光剂量荧光曲线。讨论了成像条件与蛋白形态对激活特性的影响,观察到高亮荧光蛋白在纯化形态与细胞形态下激活对比度的显著差别,说明了表征高亮荧光蛋白在细胞环境中特性的重要性。(3)探讨了利用光强依赖的光激活特性参数精确计算光激活定位显微成像激活时间从而提高成像时间分辨率的可能性,得到了光激活定位显微成像激活光强时间和强度在激活过程中的计算方法,并通过实验进行了验证。在此基础上,将这种优化方法推广到更一般的光激活荧光成像中,实现了对一般光激活荧光成像中高亮荧光蛋白激活过程的设计与调控方法。更多还原

【Abstract】 Optical microscopy is an essential tool for life science research. However due to the diffraction of light, optical microscopes are not capable of imaging biological smaller than 200 nm. The recently reported super resolution microscopic imaging techniques have break though this limit and achieved resolution up to 20 nm. One type of these techniques is Photoacitvated Localization Microscopy (PALM), this technique includes new activation and imaging light path, highly sensitive imaging camera, reconstruction algorithm and a newly reported fluorescent tags—the optical highlighter fluorescent proteins. These proteins experience significant spectral variance under the activation of high energy light. PALM systemwith favorable performance relies on optical highlighter fluorescent proteins with optimized activation features, yet these features are important references for selecting and properly controlling the activation process in practical imaging experiments. However, the study of activation behavior of the optical highlighter fluorescent proteins is significanly affected by the activation condition, this results in the diverse outcomes of activation experiments for a single protein, thus a proper parameter for profiling the activation characteristics of optical highlighter fluorescent proteins is still lacking, hence the absence of instruction for protein selecting and modulation in practical imaging experiments.Based on the characterization method of the photobleaching features of fluorescent proteins, we introduced and applied the parameters of intensity dependent photoactivation and photobleaching rates for characterizing the activation behavior of optical highlighter fluorescent proteins. By this method, we measured and explained the differences of activation rates between these proteins by the photoactivation mechanism. Moreover, by introducing the light dose concept from photodynamic therapy, we obtained the light dose dependent fluorescent expression and calculated the respective florescent curves of these proteins. At last, by studying the activation properties of the optical highlighter fluorescent protein under different activation intensities, we optimized the activation modulation of PALM imaging, the activation time is reduced by 50% without deteriorating the imaging quality.The main results of this thesis are:(1) Introduced the intensity dependent photoactivation and photobleaching rates for characterizing the activation properties of optical highlighter fluorescent proteins. Based on the characterization of the photobleaching rate of the photobleaching research, considered the effect of the primary factor of the activation imaging experiments: activation light intensity on the activation behavior of optical highlighter fluorescent proteins, this thesis introduced a new parameter of intensity dependent photoactivation and photobleaching rate, which is able to profile the inherent nature of optical highlighter fluorescent proteins under photoactivation. The experimentally obtained the intensity dependent photoactivation and photobleaching rates of four typical optical highlighter fluorescent proteins indicates that, the intensity dependent activation rates differs due to the divergences of activation mechanisms of different proteins.(2) Introduced the light dose fluorescence curve for evaluating the photoactivation properties of optical highlighter fluorescent proteins directly and quantitatively, and for straightforward referencing during the experimental photoactivation process. By experiments and theoretical calculation, we obtained the equation of the fluorescence of the optical highlighter fluorescent proteins with the photoactivation light dose during the activation process, discussed the impacts of the imaging condition and the physiological environment on the characterization of photoactivation features of optical highlighter fluorescent proteins, observed the significant difference of photoactivation contrast between the purified and physiological condition.(3) Experimentally evaluated the potential of applying the intensity dependent photoactivation rate to optimize the photoactivation process thus to increase the temporal resolution of the PALM imaging. We then extended this idea to general photoactivation fluorescent imaging techniques and achieved the design and modulation of the photoactivation process of photoactivation fluorescent imaging.更多还原