Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy.
Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy.
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DOI：
10.1021/acs.jpcb.2c07652
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发表时间：
2023-03-02
影响因子：
3.3
通讯作者：
Adams, Peter G.
中科院分区：
文献类型：
--
作者：
Meredith, Sophie A.;Kusunoki, Yuka;Connell, Simon D.;Morigaki, Kenichi;Evans, Stephen D.;Adams, Peter G.
荧光探针在生物物理研究中非常有用，可以评估生物分子的空间分布、迁移和相互作用。然而，在高浓度下，荧光团可以经历其荧光强度的“自猝灭”。更好地理解浓度猝灭效应对于避免荧光图像中的伪影和光合作用中的能量转移过程非常重要。在这里，我们展示了电泳技术可以用来控制与负载脂质双分子层（slb）相关的带电荧光团的迁移，并且可以用荧光寿命成像显微镜（FLIM）量化猝灭效应。在玻璃基板上的100 × 100 μm的围栏区域内生成了含有一定量脂联德克萨斯红（TR）荧光团的密闭slb。在脂质双分子层的平面内施加电场诱导带负电荷的tr -脂质分子向正极迁移，并在每个畜栏上产生横向浓度梯度。在FLIM图像中直接观察到TR的自猝灭，作为高浓度荧光团与其荧光寿命减少的相关性。通过改变加入slb的TR荧光团的初始浓度从0.3%到0.8% (mol/mol)，电泳时达到的最大荧光团浓度可以从2%调节到7% (mol/mol)，导致荧光寿命降低到30%，荧光强度猝灭到原来水平的10%。作为这项工作的一部分，我们展示了一种通过校正猝灭效应将荧光强度谱转换为分子浓度谱的方法。计算的浓度曲线与指数增长函数有很好的拟合，表明tr -脂质即使在高浓度下也能自由扩散。总的来说，这些发现证明了电泳在产生感兴趣分子的微尺度浓度梯度方面是有效的，并且FLIM是通过分子的光物理状态来询问分子相互作用动态变化的极好方法。
Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo “self-quenching” of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 μm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.
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影响因子：
3.4
作者：
Groves, JT;Wulfing, C;Boxer, SG
通讯作者：
Boxer, SG
DOI：
10.1016/j.bbabio.2018.06.011
发表时间：
2018-10
期刊：
Biochimica et biophysica acta. Bioenergetics
影响因子：
--
作者：
Adams PG;Vasilev C;Hunter CN;Johnson MP
通讯作者：
Johnson MP
影响因子：
4.6
作者：
Adams PG;Swingle KL;Paxton WF;Nogan JJ;Stromberg LR;Firestone MA;Mukundan H;Montaño GA
通讯作者：
Montaño GA
影响因子：
7.4
作者：
Liu, Chunming;Monson, Christopher F.;Yang, Tinglu;Pace, Hudson;Cremer, Paul S.
通讯作者：
Cremer, Paul S.
影响因子：
4.9
作者：
Bao, P.;Cartron, M. L.;Evans, S. D.
通讯作者：
Evans, S. D.