Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection.
Implementation of alternating excitation schemes in a biochip-reader for quasi-simultaneous multi-color single-molecule detection.
复制标题
在生物芯片读取器中实施交替激发方案以实现准同时多色单分子检测。
DOI：
10.1016/j.bios.2008.02.019
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发表时间：
2008
影响因子：
12.6
通讯作者：
G. Schütz
中科院分区：
文献类型：
--
作者：
C. Hesch;J. Hesse;G. Schütz
我们在这里报告的单分子生物芯片读出装置的发展，使用快速交替激发。该技术通过在激发中提供额外的颜色通道来扩展标准成像细胞仪。为了能够研究移动的物体，例如活细胞中主动运输的囊泡或脂质双层中自由扩散的脂质，照明脉冲的频率被选择为足够高以在生物分子移动通过照明区域时几乎冻结生物分子的运动。样品照明、扫描和线阵相机读出的同步产生覆盖相同样品区域的两个准同时记录的图像。通过扫描固定化的100 nm荧光乳胶珠，显示了对低至10 nm的点光源的衍射极限分辨率和高定位精度。超灵敏度通过成像单个荧光链霉亲和素分子在流体脂质双层中扩散来证明。用Cy 3和Cy 5标记的双色链霉亲和素可以很容易地在两个各自的激发通道中识别;染料位置的高度一致性证实了移动物体的共定位研究的适用性。最后，在大量的活细胞中的抗体-受体相互作用的扫描说明了这种方法用于生物芯片应用的可行性。
We report here the development of a device for single-molecule biochip readout using fast alternating excitation. The technology extends standard imaging cytometry by offering additional color channels in excitation. To enable the study of mobile objects, e.g. actively transported vesicles in living cells or freely diffusing lipids in a lipid bilayer, the frequency of the illumination pulses was chosen high enough to virtually freeze the motion of the biomolecules, as they are shifted through the illuminated area. The synchronization of sample illumination, scanning and line-camera readout yield two quasi-simultaneously recorded images covering the same sample region. Diffraction-limited resolution and high localization precision for point-light sources down to ∼10nm was shown by scanning immobilized 100nm fluorescence latex beads. Ultra-sensitivity was demonstrated by imaging single fluorescent streptavidin molecules diffusing in a fluid lipid bilayer. Two-color streptavidin labeled with Cy3 and Cy5 could be easily identified in the two respective excitation channels; high accordance in the dye positions confirms the applicability for colocalization studies of moving objects. Finally, scans of antibody–receptor interactions in large populations of live cells illustrate the feasibility of this method for biochip application.
影响因子：
3.4
作者：
Thompson, RE;Larson, DR;Webb, WW
通讯作者：
Webb, WW