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Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes
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Tethered Bichromophoric Fluorophore Quencher Voltage Sensitive Dyes
Ping Yan1, Corey D Acker1, Leslie M Loew1
1Richard D. Berlin Center for Cell Analysis and Modeling , University of Connecticut Health Center , Farmington , Connecticut 06030 , United States.
ACS Sensors
|November 27, 2018
Summary
New tethered bichromophoric fluorophore quencher (TBFQ) dyes significantly enhance voltage-sensitive dye (VSD) sensitivity for drug screening and electrical imaging. This breakthrough offers an order of magnitude improvement over existing VSDs, paving the way for better cellular monitoring.
Area of Science:
- Biophysics
- Chemical Biology
- Neuroscience Tools
Background:
- Voltage-sensitive dyes (VSDs) are crucial for in vitro drug screening and imaging electrical activity in tissues.
- Current VSDs face limitations in sensitivity, fluorescence quantum yield, and response kinetics, hindering broad application.
- Existing two-component VSD systems are limited by low quencher concentrations needed to avoid pharmacological effects.
Purpose of the Study:
- To develop novel voltage-sensitive dyes with enhanced sensitivity and concentration independence.
- To investigate tethered bichromophoric fluorophore quencher (TBFQ) dyes as a solution to VSD limitations.
- To synthesize and characterize new TBFQ dyes for improved electrical activity monitoring.
Main Methods:
- Synthesized 13 tethered bichromophoric fluorophore quencher (TBFQ) dyes, linking aminonaphthylethenylpyridinium (ANEP) fluorophores to dipicrylamine (DPA) quenchers via a hydrophobic chain.
- Tested TBFQ dyes in an artificial lipid bilayer apparatus to measure voltage-dependent fluorescence changes.
- Evaluated sensitivity (fluorescence change per mV) and response kinetics (ms range).
Main Results:
- The best TBFQ dye (TBFQ1) demonstrated a 2.5-fold fluorescence change per 100 mV, an order of magnitude improvement over conventional VSDs.
- TBFQ dyes exhibited concentration-independent sensitivity, overcoming a key limitation of previous systems.
- Response kinetics were in the 10-20 ms range, suitable for many biological applications, though fluorescence quantum yield was low (1.6%).
Conclusions:
- Tethered bichromophoric fluorophore quencher (TBFQ) dyes represent a significant advancement in voltage-sensitive dye technology.
- The TBFQ design overcomes sensitivity limitations and concentration dependence issues of prior VSDs.
- While first-generation TBFQ dyes have limitations for in vivo imaging, the design principles offer a foundation for future, more practical VSDs.
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