小蛋白,大功能!一種可用於超高分辨顯微成像的新型螢光蛋白

2015年3月3日訊 /生物谷BIOON/--最近中科院生物物理所的徐平勇課題組在著名期刊ACS NANO上發表了題為Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI)的文章，報告了一種可用作高速活細胞超解析度顯微成像的新型反覆光激活綠色螢光蛋白kylan-S。

綠色螢光蛋白(GFP)的發明因其能夠提供對於活細胞和活體動物的靶向基因修飾標記而獲得2008年諾貝爾化學獎。進一步，由基因改造的光激活螢光蛋白(PAGFP)能夠提供單分子特性，而實現了超分辨顯微成像，使得這一技術獲得2014年諾貝爾化學獎。

隨後，超高分辨顯微成像技術的發展向著活細胞動態超高時空解析度顯微成像邁進。其中，光學波動超高分辨成像技術(SOFI)是一種簡單可行的活細胞超高分辨顯微成像新技術，它利用特殊螢光蛋白（或者染料）的反覆光開關特性以及成像像素點隨時間的波動和相關特性進行成像，因其可以突破單分子發光限制，同時實現多個螢光分子高速成像而迅速成為國際關注的熱點。

kylan-S與現有的適用於SOFI成像的Dronpa相比具有突出的優點：在波動（fluctuation）狀態下的螢光亮度比Dronpa高6-8倍，波動動態範圍高4倍，同時具有極高的光學穩定性。Skylan-S是一種單體，因此能夠用於活細胞成像而不影響目標蛋白的定位和功能。基於這些特性，Skylan-S應用於SOFI超分辨成像中具有極高的時空解析度。在SOFI成像中，獲得了1.5秒的超高時空解析度成像（每幀3毫秒，500幀進行SOFI重構），可利用4階SOFI獲得clathrin-coated pits（CCP）的環狀結構結構，空間解析度好於100nm。

同時，Skylan-S能夠動態觀察亞細胞結構60s以上。而Dronpa在第一幀就無法實現這一高解析度，在30s中超過一半基本被漂白。利用Skylan-S和Dronpa分別標記細胞的肌動蛋白絲，Skylan-S能夠以豐富灰階顯示不同標記濃度的蛋白絲的精細結構，而Dronpa則僅能提供信噪比非常有限的圖像。另外，Skylan-S在較高能量的488納米雷射照射下具有很好的單分子特性，適用於PALM成像。同時，它也廣泛適用於傳統的共聚焦和雙光子成像。（生物谷Bioon.com）

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ACS NANO  DOI: 10.1021/nn5064387

Development of a Reversibly Switchable Fluorescent Protein for Super-Resolution Optical Fluctuation Imaging (SOFI)

Xi Zhang, Xuanze Chen, Zhiping Zeng, Mingshu Zhang, Yujie Sun, Peng Xi, Jianxin Peng, and Pingyong Xu

Reversibly switchable fluorescent proteins (RSFPs) can be effectively used for super-resolution optical fluctuation imaging (SOFI) based on the switching and fluctuation of single molecules. Several properties of RSFPs strongly influence the quality of SOFI images. These properties include (i) the averaged fluorescence intensity in the fluctuation state, (ii) the on/off contrast ratio, (iii) the photostability, and (iv) the oligomerization tendency. The first three properties determine the fluctuation range of the imaged pixels and the SOFI signal, which are of essential importance to the spatial resolution, and the last may lead to artificial aggregation of target proteins. The RSFPs that are currently used for SOFI are low in averaged fluorescence intensity in the fluctuation state, photostability, and on/off contrast ratio, thereby limiting the range of application of SOFI in biological super-resolution imaging. In this study, we developed a novel monomeric green RSFP termed Skylan-S, which features very high photostability, contrast ratio, and averaged fluorescence intensity in the fluctuation state. Taking advantage of the excellent optical properties of Skylan-S, a 4-fold improvement in the fluctuation range of the imaged pixels and higher SOFI resolution can be obtained compared with Dronpa. Furthermore, super-resolution imaging of the actin or tubulin structures and clathrin-coated pits (CCPs) in living U2OS cells labeled with Skylan-S was demonstrated using the SOFI technique. Overall, Skylan-S developed with outstanding photochemical properties is promising for long-time SOFI imaging with high spatial-temporal resolution.