本文選題：超分辨成像 + 細胞穿膜肽　； 參考：《華中科技大學》2015年博士論文

【摘要】：由于存在光的衍射極限,光學成像一直以來被認為無法突破衍射極限而獲取更高的空間分辨率。近年來出現(xiàn)的超分辨成像技術(shù)打破了衍射極限,將可分辨的空間尺度提高到了250納米以下,并因此獲得了2014年的諾貝爾化學獎。 單分子定位超分辨成像是超分辨成像的一種方式。其成像過程不需要依賴復雜昂貴的光學儀器,但需要光可調(diào)的熒光探針來實現(xiàn)超分辨成像。在目前的熒光探針中,有機熒光探針由于其所包含的熒光染料具有優(yōu)秀的光物理性質(zhì),在活細胞超分辨成像中具有獲取更高時間及空間分辨率的潛在優(yōu)勢。然而,目前成功用于活細胞超分辨成像的有機熒光探針種類非常有限,嚴重阻礙了超分辨成像的進一步發(fā)展。 本文提出了一種構(gòu)建新型透膜有機熒光探針的通用策略,并實現(xiàn)了活細胞超分辨成像。該策略將細胞穿膜肽(rR)3R2引入到熒光探針構(gòu)建中,利用細胞穿膜肽能夠攜帶貨物分子穿透細胞膜的作用,將光可調(diào)的熒光染料及能夠特異性標記的識別基團攜帶進入到活細胞內(nèi),實現(xiàn)了特異性標記及超分辨成像。本文以絲狀肌動蛋白及半胱氨酸組織蛋白酶為特異性標記目標,實現(xiàn)了在活細胞內(nèi)對內(nèi)源性絲狀肌動蛋白及溶酶體細胞器的超分辨成像。具體研究內(nèi)容如下： (1)設(shè)計并合成了針對活細胞內(nèi)源性絲狀肌動蛋白的透膜有機熒光探針,包括含有普通熒光染料(RhB、FITC)的探針及光激活熒光染料(PA-RhB)的探針。 (2)首先用所合成的含有普通熒光染料的探針研究了此類探針的活細胞成像結(jié)果,包括：不同孵育濃度、不同孵育時間,以及熒光染料對標記特異性的影響。根據(jù)上述步驟,優(yōu)化確定出了此類探針對于絲狀肌動蛋白標記的最佳實驗條件。 (3)在(2)的研究基礎(chǔ)上進一步研究了含有不同有機熒光染料(RhB、FITC、 PA-RhB)的探針在超分辨成像中的實驗條件,包括：探針的光穩(wěn)定及光激活性質(zhì)、重建算法、曝光時間、激發(fā)光強及重建幀數(shù)。最終,本文利用所構(gòu)建的含有光激活熒光染料PA-RhB的探針國際上首次實現(xiàn)了活細胞內(nèi)源性絲狀肌動蛋白的超分辨成像,并發(fā)現(xiàn)此探針具有免激活、耐漂白的獨特性質(zhì)。在此基礎(chǔ)上,本文國際上首次成功地記錄了絲狀肌動蛋白的重排動力學過程,獲得了84納米的空間分辨率及10秒的時間分辨率。 (4)本文利用包含光激活熒光染料Caged-Rh110并能特異性標記溶酶體的透膜有機熒光探針,實現(xiàn)了活細胞內(nèi)溶酶體的超分辨成像,獲得了64納米的空間分辨率及1.2-2秒的時間分辨率。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：Q2-33

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