一种活细胞超分辨成像有机荧光探针构建新策略

发布时间：2018-05-12 04:19

本文选题：超分辨成像 + 细胞穿膜肽　；参考：《华中科技大学》2015年博士论文

【摘要】：由于存在光的衍射极限,光学成像一直以来被认为无法突破衍射极限而获取更高的空间分辨率。近年来出现的超分辨成像技术打破了衍射极限,将可分辨的空间尺度提高到了250纳米以下,并因此获得了2014年的诺贝尔化学奖。单分子定位超分辨成像是超分辨成像的一种方式。其成像过程不需要依赖复杂昂贵的光学仪器,但需要光可调的荧光探针来实现超分辨成像。在目前的荧光探针中,有机荧光探针由于其所包含的荧光染料具有优秀的光物理性质,在活细胞超分辨成像中具有获取更高时间及空间分辨率的潜在优势。然而,目前成功用于活细胞超分辨成像的有机荧光探针种类非常有限,严重阻碍了超分辨成像的进一步发展。本文提出了一种构建新型透膜有机荧光探针的通用策略,并实现了活细胞超分辨成像。该策略将细胞穿膜肽(rR)3R2引入到荧光探针构建中,利用细胞穿膜肽能够携带货物分子穿透细胞膜的作用,将光可调的荧光染料及能够特异性标记的识别基团携带进入到活细胞内,实现了特异性标记及超分辨成像。本文以丝状肌动蛋白及半胱氨酸组织蛋白酶为特异性标记目标,实现了在活细胞内对内源性丝状肌动蛋白及溶酶体细胞器的超分辨成像。具体研究内容如下： (1)设计并合成了针对活细胞内源性丝状肌动蛋白的透膜有机荧光探针,包括含有普通荧光染料(RhB、FITC)的探针及光激活荧光染料(PA-RhB)的探针。 (2)首先用所合成的含有普通荧光染料的探针研究了此类探针的活细胞成像结果,包括：不同孵育浓度、不同孵育时间,以及荧光染料对标记特异性的影响。根据上述步骤,优化确定出了此类探针对于丝状肌动蛋白标记的最佳实验条件。 (3)在(2)的研究基础上进一步研究了含有不同有机荧光染料(RhB、FITC、 PA-RhB)的探针在超分辨成像中的实验条件,包括：探针的光稳定及光激活性质、重建算法、曝光时间、激发光强及重建帧数。最终,本文利用所构建的含有光激活荧光染料PA-RhB的探针国际上首次实现了活细胞内源性丝状肌动蛋白的超分辨成像,并发现此探针具有免激活、耐漂白的独特性质。在此基础上,本文国际上首次成功地记录了丝状肌动蛋白的重排动力学过程,获得了84纳米的空间分辨率及10秒的时间分辨率。 (4)本文利用包含光激活荧光染料Caged-Rh110并能特异性标记溶酶体的透膜有机荧光探针,实现了活细胞内溶酶体的超分辨成像,获得了64纳米的空间分辨率及1.2-2秒的时间分辨率。
[Abstract]:Because of the diffraction limit of light, optical imaging has been thought to be unable to break through the diffraction limit and obtain higher spatial resolution. The super-resolution imaging techniques that have emerged in recent years have broken the diffraction limit by raising the resolution spatial scale to below 250 nanometers and winning the 2014 Nobel Prize in Chemistry. Single molecular positioning super-resolution imaging is a way of super-resolution imaging. The imaging process does not depend on complex and expensive optical instruments, but requires a light-adjustable fluorescence probe to achieve super-resolution imaging. The organic fluorescent probes have the potential advantage of obtaining higher temporal and spatial resolution in living cell superresolution imaging because of their excellent photophysical properties. However, the successful use of organic fluorescent probes for living cell superresolution imaging is very limited, which seriously hinders the further development of super-resolution imaging. In this paper, a general strategy for constructing a novel transmembrane organic fluorescence probe is proposed, and a living cell super-resolution imaging is realized. In this strategy, the cellular transmembrane peptide rRn3R2 was introduced into the construction of fluorescent probe, and the transmembrane peptide was used to carry cargo molecules through the cell membrane, and the light-adjustable fluorescent dye and the specific labeled recognition group were carried into the living cells. Special labeling and super-resolution imaging were realized. Using filamentous actin and cysteine cathepsin as specific labeling targets, superresolution imaging of endogenous filamentous actin and lysosomal organelles in living cells has been achieved. The specific contents of the study are as follows: 1) the transmembrane organic fluorescent probes for endogenous filamentous actin in living cells were designed and synthesized, including those containing the ordinary fluorescent dye RhBFITCand the photoactivated dye PA-RhB. (2) the living cell imaging results of the probes containing common fluorescent dyes were studied firstly, including: different incubation concentration, different incubation time, and the effect of fluorescent dyes on the labeling specificity. According to the above steps, the optimal conditions for the labeling of filamentous actin with this kind of probe were optimized. The experimental conditions of the probe containing different organic fluorescent dyes RhBFITC (PA-RhB) in super-resolution imaging are further studied, including: the photostability and photoactivation of the probe, the reconstruction algorithm, and the exposure time. Stimulated light intensity and reconstructed frame count. Finally, the super-resolution imaging of endogenous filamentous actin in living cells has been realized for the first time in the world using a probe containing photoactivated fluorescent dye PA-RhB, and it is found that the probe has the unique properties of non-activation and bleach resistance. On this basis, the dynamic process of filamentous actin rearrangement has been successfully recorded for the first time in the world, and the spatial resolution of 84 nanometers and the temporal resolution of 10 seconds have been obtained. In this paper, the transmembrane organic fluorescence probe containing photoactivated fluorescent dye Caged-Rh110 and specific labelling of lysosomes was used to realize the super-resolution imaging of lysosomes in living cells. The spatial resolution of 64 nanometers and the temporal resolution of 1.2-2 seconds were obtained.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q2-33

【相似文献】