Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules
Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules
复制标题
FastFLIM 可调谐 PIE 和同步门控检测，用于单分子快速动态的定量显微镜测量
DOI：
10.1117/12.2217434
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
S. Liao
中科院分区：
文献类型：
--
作者：
Yuansheng Sun;U. Coskun;A. C. Ferreon;B. Barbieri;S. Liao
两种荧光物质之间的串扰给荧光显微成像带来了问题，特别是在共定位、Förster共振能量转移(FRET)、荧光互相关光谱(FCCS)等定量测量中。在激光扫描共焦显微镜中，激光器可以通过声光可调谐滤光器(AOTF)在微秒级进行交替行扫描来开启和关闭激光器，以避免串扰，同时最小化同一像素位置上两个激光器之间的时延。相比之下，脉冲交错激发(PIE)技术在纳秒范围内同步两个不同波长的脉冲激光，从而能够同时且定量地测量两个荧光物种的超快动力学，而不会造成串扰污染。这一功能对于许多细胞生物学应用至关重要，例如，在研究蛋白质-蛋白质相互作用或细胞信号事件的FRET测量中准确确定化学计量比，通过消除串扰造成的虚假交叉相关来检测FCC中较弱的结合。这块馅饼已经与时间相关单光子计数(TCSPC)电子设备一起使用。在这里，我们描述了一种使用数字频域(DFD)技术的新型PIE开发--FastFLIM，它提供了可调的PIE设置和同步门控检测，根据特定应用进行了定制和优化。文中还介绍了FastFLIM的几种饼图设置和测量实例。结合ALBA和Q2系统的灵敏度，PIE使我们能够定量测量单分子的快速动力学。
The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique — FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.
登录
查看更多内容
影响因子：
3.4
作者：
Lee, NK;Kapanidis, AN;Weiss, S
通讯作者：
Weiss, S
影响因子：
7.7
作者：
Clegg, Robert M.
通讯作者：
Clegg, Robert M.
影响因子：
46.2
作者：
Day RN;Davidson MW
通讯作者：
Davidson MW
影响因子：
35
作者：
通讯作者：
--