Single molecule localization imaging of telomeres and centromeres using fluorescence in situ hybridization and semiconductor quantum dots
Single molecule localization imaging of telomeres and centromeres using fluorescence in situ hybridization and semiconductor quantum dots
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使用荧光原位杂交和半导体量子点对端粒和着丝粒进行单分子定位成像
DOI：
10.1088/1361-6528/aabf72
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发表时间：
2018
期刊：
影响因子：
3.5
通讯作者：
Yiping Cui
中科院分区：
文献类型：
--
作者：
Le Wang;Shenfei Zong;Zhuyuan Wang;Ju Lu;Chen Chen;Ruohu Zhang;Yiping Cui
单分子定位显微镜(SMLM)是在纳米尺度对生物靶标成像的有力工具。在这份报告中，我们介绍了使用荧光原位杂交(FISH)对端粒和着丝粒的SMLM成像。用端粒或着丝粒互补DNA链修饰CdSSe/ZnS量子点，制备FISH探针。使用有机荧光标记的商业肽核酸(PNA)探针的SMLM成像实验也证明了使用量子点FISH探针的优势。与PNA探针相比，量子点探针具有以下优点。首先，量子点的荧光闪烁可以在水溶液或不含硫醇的PBS缓冲液中实现，硫醇是有机荧光团闪烁的关键缓冲成分。其次，量子点探针的荧光闪烁只需要一个激发光(即405 nm)。而有机荧光团的荧光闪烁通常需要两个照明光，即激活光(即405 nm)和成像光。第三，高的量子产率、多次开关次数和良好的光学稳定性使得量子点更适合于长期成像。在端粒和着丝粒成像实验中获得的定位精度约为30 nm，远远超过了衍射极限。SMLM使人们能够在分子水平上对端粒或着丝粒有新的了解，甚至有可能确定端粒的长度，成为一种潜在的端粒相关研究技术。
Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores’ blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.