FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope
FRET efficiency measurement in a molecular tension probe with a low-cost frequency-domain fluorescence lifetime imaging microscope
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DOI：
10.1117/1.jbo.24.12.126501
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发表时间：
2019-12-01
影响因子：
3.5
通讯作者：
Boustany, Nada N.
中科院分区：
文献类型：
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作者：
Dumas, John-Paul;Jiang, James Y.;Boustany, Nada N.
我们证明了用低成本的频域荧光寿命成像显微镜（FD-FLIM）测量FRET效率的可能性。该系统采用单频调制激励，可使用具有成本效益的激光源和电子设备，简化数据采集和分析，并具有双通道检测能力。在用香豆素6校准后，我们测量了在活细胞中表达的mTFP 1-mVenus FRET标准品中的表观供体寿命。我们通过区分分别对应于已知标准品的高和低FRET效率的mTFP 1的短和长寿命来评估系统的灵敏度。此外，我们发现黏着斑张力传感器VinTS在粘着斑处的寿命（2.30 +/- 0.16 ns）显著（p < 10（-6））长于未加载TSMod探针的寿命（2.02 +/- 0.16 ns）。表达FRET标准品的样品的像素停留时间为6.8 μ s，信号通常比VinTS高一个数量级。从测量的表观寿命计算的标准品的表观FRET效率（E-FrET（app））与其已知的FRET效率线性相关，系数为0.92至0.99（R-2 = 0.98）。这种关系作为一个校准曲线转换表观FRET真正的FRET和规避需要测量多指数寿命衰减。对于VinTS，这种方法产生了18%至19.5%的FRET效率，与公布的值一致。总之，我们的研究结果表明，具有成本效益，快速，灵敏的FD-FLIM方法，有可能促进FLIM在机械生物学和FRET为基础的生物传感的应用。(C)作者。由SPIE在知识共享署名4.0未移植许可下发布。
We demonstrate the possibility of measuring FRET efficiency with a low-cost frequency-domain fluorescence lifetime imaging microscope (FD-FLIM). The system utilizes single-frequency-modulated excitation, which enables the use of cost-effective laser sources and electronics, simplification of data acquisition and analysis, and a dual-channel detection capability. Following calibration with coumarin 6, we measured the apparent donor lifetime in mTFP1-mVenus FRET standards expressed in living cells. We evaluated the system's sensitivity by differentiating the short and long lifetimes of mTFP1 corresponding to the known standards' high and low FRET efficiency, respectively. Furthermore, we show that the lifetime of the vinculin tension sensor, VinTS, at focal adhesions (2.30 +/- 0.16 ns) is significantly (p < 10(-6)) longer than the lifetime of the unloaded TSMod probe (2.02 +/- 0.16 ns). The pixel dwell time was 6.8 mu s for samples expressing the FRET standards, with signal typically an order of magnitude higher than VinTS. The apparent FRET efficiency (E-FrET(app)) of the standards, calculated from the measured apparent lifetime, was linearly related to their known FRET efficiency by a factor of 0.92 to 0.99 (R-2 = 0.98). This relationship serves as a calibration curve to convert apparent FRET to true FRET and circumvent the need to measure multiexponential lifetime decays. This approach yielded a FRET efficiency of 18% to 19.5%, for VinTS, in agreement with published values. Taken together, our results demonstrate a cost-effective, fast, and sensitive FD-FLIM approach with the potential to facilitate applications of FLIM in mechanobiology and FRET-based biosensing. (C) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License.