Exploring the self-quenching of fluorescent probes incorporated into model lipid membranes using electrophoresis and fluorescence lifetime imaging microscopy

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 × 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.