在活细胞中超分辨率成像的可光激活探头
Elias A Halabi1, Zacharias Thiel1, Nils Trapp1
1Laboratory of Organic Chemistry, ETH Zurich , Vladimir-Prelog-Weg 3, CH-8093 Zurich, Switzerland.
Journal of the American Chemical Society
|August 19, 2017
概括
研究人员开发了一种用于单个分子水平上检测酶活性的新型探针. 这种化探测器能够对活细胞中的酶活性进行超高分辨率成像,为酶的定位和功能提供了新的见解.
科学领域:
- 化学生物学
- 分子成像
- 酵素学
背景情况:
- 在生物过程中,雌激酶活动至关重要,但其精确的定位和动态难以研究.
- 现有的方法往往缺乏在单个分子水平上可视化酶活性的灵敏度和分辨率.
研究的目的:
- 开发一种可双激活的化探针,以检测单分子分辨率的雌激酶活性.
- 为了使活细胞中的酶活性能够超分辨率成像.
- 为了证明探测器在研究酶抑制剂中的有用性.
主要方法:
- 开发一种基于diazoindanone的化探针.
- 使用沃尔夫重组在酶预激活时生成信号.
- 使用单分子定位显微镜 (SMLM) 进行超分辨率成像.
- 活细胞成像和溶液研究以验证探针的性能和选择性.
主要成果:
- 该探针具有双重激活性,仅在经过酶预激活和光照射后产生光色素染料.
- 由于与细胞大分子的共价结合,光活性染料保持局部,防止扩散.
- SMLM成功地重建了酶活动的超高分辨率图像.
- 抑制人类的碳酸酶I和II显著改变了检测到的信号的数量和分布.
结论:
- 为单个分子检测酶活性建立了一个新的传感机制.
- 开发的探测器提供了前所未有的空间分辨率来可视化活细胞中的酶活动.
- 这种方法有可能研究各种酶点及其在生物系统中的作用.
相关概念视频
Super-resolution Fluorescence Microscopy
14.6K
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
14.6K
Protein Dynamics in Living Cells
2.8K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
2.8K
Labeling DNA Probes
9.6K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
9.6K


