FlAsH-PALM: super-resolution pointillist imaging with FlAsH-tetracysteine labeling

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Super-resolution microscopy, such as photoactivated localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM), enables imaging below the diffraction limit.
• Conventional labeling methods (antibodies, protein fusions) can disrupt protein function and distribution.
• The FlAsH labeling technique offers improved preservation of protein function and compatibility with live-cell imaging.

Purpose of the Study:

• To present a detailed protocol for applying FlAsH-PALM (fluorescence self-labeling with a tetracysteine tag and photoactivated localization microscopy) for high-resolution protein imaging.
• To demonstrate the utility of FlAsH-PALM for visualizing HIV integrase in viral particles and infected cells.
• To facilitate the adoption of FlAsH-PALM for imaging other proteins and biological structures.

Main Methods:

• Application of the FlAsH labeling technique for specific protein tagging.
• Utilizing single-molecule localization microscopy (SMLM) principles, specifically PALM, for super-resolution imaging.
• Imaging HIV integrase in isolated virions and infected cells.

Main Results:

• Achieved sub-diffraction resolution imaging of HIV integrase spatial distribution.
• Obtained high-resolution images of HIV morphology without affecting viral replication.
• Demonstrated the potential of FlAsH-PALM for imaging delicate proteins in intracellular compartments.

Conclusions:

• FlAsH-PALM is a valuable super-resolution technique that minimizes disruption to protein function and cellular processes.
• The detailed protocol enables broader application of FlAsH-PALM for high-resolution imaging of various biological targets.
• This method holds promise for advancing our understanding of protein organization and viral dynamics at the nanoscale.