Imaging Membrane Proteins Using Total Internal Reflection Fluorescence Microscopy (TIRFM) in Mammalian Cells

Area of Science:

• Cell Biology
• Biophysics
• Microscopy Techniques

Background:

• The cell surfaceome is crucial in biological processes and disease.
• Precisely identifying cell surface proteins and their regulation is challenging.
• Total Internal Reflection Fluorescence Microscopy (TIRFM) offers high precision for cell membrane studies.

Purpose of the Study:

• To present a protocol for micromirror TIRFM analysis.
• To investigate the translocation of optogenetically activated protein kinase C-ε.
• To demonstrate the utility of TIRFM for live-cell imaging of cell surface proteins.

Main Methods:

• Utilized micromirror TIRFM for enhanced spatial resolution.
• Employed optogenetics to activate protein kinase C-ε.
• Analyzed protein translocation dynamics in HEK293-T cells.

Main Results:

• Successfully implemented micromirror TIRFM for live-cell imaging.
• Demonstrated the translocation of protein kinase C-ε to the cell surface upon optogenetic activation.
• Achieved high signal-to-noise ratio for precise protein localization.

Conclusions:

• Micromirror TIRFM is a powerful technique for studying cell surface protein dynamics.
• Optogenetic activation provides precise control over protein localization studies.
• This protocol facilitates detailed investigation of cell surfaceome regulation.