Imaging Fluorescence Blinking of a Mitochondrial Localization Probe: Cellular Localization Probes Turned into...

Area of Science:

• Biophysics
• Cell Biology
• Biochemistry

Background:

• Mitochondrial membranes are crucial for cellular metabolism but challenging to study in live cells.
• Existing probes often lack the ability to provide multiparametric information about the membrane microenvironment.
• 10-nonyl acridine orange (NAO) is a common fluorophore for mitochondrial membranes.

Purpose of the Study:

• To develop a strategy for transforming the NAO fluorophore into a multifunctional probe of mitochondrial membrane microenvironments.
• To investigate the blinking kinetics of NAO and their sensitivity to various environmental factors.
• To demonstrate the applicability of this blinking analysis in live cells.

Main Methods:

• Transient State (TRAST) studies of NAO in small unilamellar vesicles (SUVs).
• Computational simulations to understand NAO photophysics.
• TRAST imaging of NAO blinking kinetics in live cells.

Main Results:

• NAO exhibits environment-sensitive blinking kinetics, reflecting oxygen concentration, redox state, membrane charge, fluidity, and lipid composition.
• Cardiolipin concentration and acyl chain composition significantly influence NAO blinking.
• Blinking kinetics are coupled to proton transfer events, local pH, and membrane buffering properties.
• Spatially resolved imaging of NAO blinking in live cells is achievable.

Conclusions:

• NAO blinking kinetics can be used to probe diverse mitochondrial membrane microenvironmental parameters.
• This strategy transforms a conventional fluorophore into a multiparametric sensor.
• The method offers new possibilities for fundamental studies of lipid vesicles and live cell membranes.