A Photoswitchable Solvatochromic Dye for Probing Membrane Ordering by RESOLFT Super-resolution Microscopy

Area of Science:

• Membrane biophysics
• Fluorescence microscopy
• Chemical biology

Background:

• Understanding lipid organization in biological membranes is crucial for cellular function.
• Current imaging techniques often lack the resolution to discern fine membrane structures.
• Developing tools for super-resolution imaging of membrane dynamics is an active research area.

Purpose of the Study:

• To develop a switchable fluorescent probe for mapping lipid ordering in vesicle membranes.
• To achieve imaging resolution beyond the diffraction limit using light-controlled fluorescence.
• To investigate membrane polarity and distinguish lipid domains within synthetic vesicles.

Main Methods:

• A fluorescent dyad combining Nile Red and a spironaphthoxazine quencher was synthesized.
• Photoswitching and Förster Resonance Energy Transfer (FRET) quenching controlled fluorescence.
• Synthetic lipid vesicles were imaged using confocal microscopy and the developed probe.
• Ratiometric detection was employed to assess membrane polarity and lipid ordering.

Main Results:

• The switchable fluorescent dyad enabled imaging of lipid ordering with enhanced resolution (average 2.5-fold).
• Visible light controlled the probe's fluorescence via photoswitching and FRET.
• Membrane polarity was successfully probed using ratiometric detection.
• Distinct domains with different lipid ordering were resolved within the same vesicle membrane.

Conclusions:

• A novel light-controlled fluorescent dyad provides super-resolution mapping of lipid ordering in membranes.
• The developed probe offers a valuable tool for studying membrane heterogeneity and dynamics.
• This approach advances the capability to visualize nanoscale membrane structures and functions.