Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored 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 i...
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Published in | The journal of physical chemistry. B Vol. 127; no. 8; pp. 1715 - 1727 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
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American Chemical Society
02.03.2023
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Abstract | 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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AbstractList | 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. 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. |
Author | Meredith, Sophie A. Kusunoki, Yuka Connell, Simon D. Evans, Stephen D. Morigaki, Kenichi Adams, Peter G. |
AuthorAffiliation | University of Leeds Astbury Centre for Structural Molecular Biology School of Physics and Astronomy Graduate School of Agricultural Science Graduate School of Agricultural Science and Biosignal Research Center |
AuthorAffiliation_xml | – name: Graduate School of Agricultural Science – name: Astbury Centre for Structural Molecular Biology – name: School of Physics and Astronomy – name: University of Leeds – name: Graduate School of Agricultural Science and Biosignal Research Center |
Author_xml | – sequence: 1 givenname: Sophie A. surname: Meredith fullname: Meredith, Sophie A. organization: University of Leeds – sequence: 2 givenname: Yuka surname: Kusunoki fullname: Kusunoki, Yuka organization: Graduate School of Agricultural Science – sequence: 3 givenname: Simon D. orcidid: 0000-0003-2500-5724 surname: Connell fullname: Connell, Simon D. organization: University of Leeds – sequence: 4 givenname: Kenichi orcidid: 0000-0002-2454-6513 surname: Morigaki fullname: Morigaki, Kenichi organization: Graduate School of Agricultural Science and Biosignal Research Center – sequence: 5 givenname: Stephen D. orcidid: 0000-0001-8342-5335 surname: Evans fullname: Evans, Stephen D. organization: University of Leeds – sequence: 6 givenname: Peter G. orcidid: 0000-0002-3940-8770 surname: Adams fullname: Adams, Peter G. email: p.g.adams@leeds.ac.uk organization: University of Leeds |
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Snippet | Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can... Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can... |
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SubjectTerms | B: Biophysical and Biochemical Systems and Processes Electrophoresis Fluorescent Dyes Lipid Bilayers - chemistry Membranes Microscopy, Fluorescence - methods |
Title | Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy |
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