Exploring the self-quenching of fluorescent probes incorporated into model lipid membranes using electrophoresis and fluorescence lifetime imaging microscopy
Exploring the self-quenching of fluorescent probes incorporated into model lipid membranes using electrophoresis and fluorescence lifetime imaging microscopy
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使用电泳和荧光寿命成像显微镜探索掺入模型脂质膜中的荧光探针的自猝灭
DOI：
10.1016/j.bpj.2022.11.1323
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发表时间：
2023
影响因子：
3.4
通讯作者：
Meredith S
中科院分区：
文献类型：
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作者：
Meredith S
荧光探针在生物物理研究中用于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非常重要，并且与光合作用中的能量转移过程有关。在这里，我们展示了电泳技术可以用来控制负载脂质双层（slb）内带电荧光团的迁移，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个Â 100 μm的圈区内生成了含有一定量德克萨斯红（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slb中TR荧光团的初始浓度从0.3%到0.8%（摩尔/摩尔），电泳过程中达到的最大荧光团浓度可以从2%到7%（摩尔/摩尔）调节，从而导致最大猝灭率从30%到70%。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。计算的浓度曲线的形状揭示了在高堆积密度下脂质-脂质相互作用的微妙证据。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 artefacts in fluorescence images and is relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores within supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of Texas Red (TR) fluorophores were generated within 100 Â 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 molecules towards 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%(mole/mole), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7%(mole/mole), leading to maximal quenching of 30% up to 70%. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The shape of the calculated concentration profiles revealed evidence of subtle lipid-lipid interactions at high packing densities. 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.