生物大分子国家重点实验室,生命科学仪器研制中心
国家自然科学基金 (81427802 , 31400658,31270884,30900268),科技部重点研发计划(2016YFA0502400),北京市自然科学基金(5122026, 5092017),中国科学院青年创新促进会(2011087)
National Laboratory of Biomacromolecules, Institute of Biophysics,Center for Biological Instrument Development
This work was supported by a grant from the National Key Research and Development Program of China (2016YFA0502400) , Natural Science Foundation of China (81427802 31270884, 30900268 and 31400658), the Beijing Natural Science Foundation (5122026, 5092017), and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2011087)
近十年来,基于单分子定位的PALM成像技术快速发展,将显微镜的分辨率提高到了2~25nm. 本文发现PALM成像过程中采用的激发光强度与成像的定位精度之间有密切的联系. 我们分别选择了PALM成像使用的光激活荧光蛋白、光转换荧光蛋白和光开关荧光蛋白中最常用的荧光蛋白进行验证. 实验发现伴随激光强度的增加,大部分荧光蛋白的光子数先升高然后趋于饱和,背景噪声几乎线性升高. 进一步分析发现荧光蛋白的定位误差随着激光强度增强先降低后升高,因此选用合适的激光强度在PALM成像实验中至关重要. 如何提高PALM成像的分辨率一直是科学家研究的热点,本研究内容可以指导研究人员在PALM成像中选用合适的激发光强度,从而得到高分辨率的图像.
PALM imaging based on single molecule localization has developed rapidly in the past decades.Using PALM, the resolution of microscope has been increased to 2-25 nm. In this study, we find that there is a close relationship between the laser power density and the localization precision in PALM imaging. To explore this relationship ,we tested the most commonly used fluorescent proteins (FPs), including photoactivatable, photoconvertable, and photoswitchable FPs in PALM imaging. The results show that with the increase of the laser power density, the photon yield of most FPs firstly increased and then became saturated, and the background noise increased almost linearly. The analysis shows that with the increasing laser power density, the localization error first decreased, and then increased. Therefore, it is very important to select the appropriate laser power density in PALM imaging. How to improving the resolution of PALM imaging has been a hot research topic. The results in this study can guide researchers to select appropriate laser power density in PALM imaging to obtain images with high resolution.
樊春燕,李媛媛,谷陆生,李尉兴,徐晓君,张淑文,王磊,陈艳,薛艳红,纪伟. PALM成像中激光强度和定位精度相关性的研究[J].生物化学与生物物理进展,2017,44(6):534-536
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