Photochromic Fluorescent Probe Strategy for the Super-resolution Imaging of Biologically Important Biomarkers

Area of Science:

• Biomedical Engineering
• Chemical Biology
• Microscopy

Background:

• Developing responsive fluorescent probes is crucial for visualizing biological processes.
• Photochromic probes offer unique optical properties but often face challenges in aqueous environments and cellular imaging.
• Human serum albumin (HSA) is a potential carrier for improving probe solubility and cell permeability.

Purpose of the Study:

• To design and characterize a β-galactosidase (β-Gal)-responsive photochromic fluorescent probe (NpG) that prebinds to HSA.
• To develop a novel imaging platform (NpG@HSA) for enhanced cellular uptake and visualization of β-Gal activity.
• To utilize the probe for high-resolution subcellular localization of β-Gal activity using STORM microscopy.

Main Methods:

• Design of a β-galactosidase-responsive photochromic fluorescent probe (NpG) incorporating a naphthalimide fluorophore and a spiropyran unit.
• Formation of a probe/protein hybrid (NpG@HSA) by prebinding NpG to human serum albumin.
• Cellular imaging experiments to visualize probe uptake and β-Gal activity using fluorescence microscopy and STORM.
• Photoisomerization studies of the merocyanine form for advanced imaging capabilities.

Main Results:

• The NpG@HSA hybrid exhibited enhanced fluorescence emission (520 nm) and improved cell permeability and solubility.
• β-Gal mediated cleavage of NpG@HSA resulted in a red fluorescence shift (620 nm) and formation of NpM@HSA.
• STORM imaging with NpG@HSA achieved nanoscale precision in determining the subcellular distribution of β-Gal activity.
• The probe demonstrated minimal phototoxicity, excellent photostability, and reversibility for advanced microscopy.

Conclusions:

• The NpG@HSA system provides a versatile imaging platform for photochromic fluorescent probes.
• This approach enables precise visualization of enzyme activity and subcellular localization of biomarkers.
• The study highlights the potential for designing advanced probes for disease-specific biomarker detection in cellular processes.