STED nanoscopy in living cells using Fluorogen Activating Proteins

Area of Science:

• Cellular imaging and microscopy
• Molecular biology and genetics
• Biophysics

Background:

• Live cell super-resolution microscopy requires specific labeling strategies.
• Genetically encoded tags offer precise protein localization for imaging.
• Malachite Green (MG) is a fluorogen with potential for advanced microscopy techniques.

Purpose of the Study:

• To evaluate a genetically encoded Malachite Green (MG) binding fluorogen activating protein (FAP) for live cell super-resolution microscopy.
• To test the efficacy of both extracellular and intracellular FAPs for stimulated emission depletion (STED) nanoscopy.
• To assess the achievable resolution using this novel labeling approach.

Main Methods:

• Development and expression of genetically encoded FAPs in living cells.
• Utilized membrane-expressed FAP and intracellular FAP-actin fusion constructs.
• Performed live cell stimulated emission depletion (STED) nanoscopy using MG fluorogens.

Main Results:

• Demonstrated the effectiveness of the genetically encoded FAP for live cell STED nanoscopy.
• Achieved observed full width at half maximum (FWHM) values of 110-122 nm for cellular structures.
• Depletion data indicated a potential instrumental resolution of 70 nm.

Conclusions:

• Genetically encoded MG-binding FAPs are effective tools for live cell super-resolution imaging.
• This approach enables visualization of subcellular structures with nanometer-scale resolution.
• The FAP system offers a promising alternative for advanced live cell nanoscopy.