Single-Molecule Localization Microscopy of Subcellular Protein Distribution in Neurons

Area of Science:

• Cell Biology
• Microscopy
• Neuroscience

Background:

• Superresolution fluorescence microscopy surpasses conventional limits (<200 nm).
• Single-molecule localization microscopy (SMLM) provides high-resolution, quantitative subcellular protein distribution analysis.
• SMLM is applicable to live cells and widely used in biological research, including neuroscience.

Purpose of the Study:

• To provide a detailed protocol for SMLM visualization of endogenous proteins in neurons.
• To extend the protocol for imaging other adherent cultured cells.
• To present methods for both live-cell and fixed-cell imaging of endogenous proteins.

Main Methods:

• Single-molecule localization microscopy (SMLM) protocol.
• Immunolabeling techniques for fixed cells.
• Intrabody use for live-cell SMLM.
• CRISPR/Cas9 for endogenous tagging.

Main Results:

• Demonstration of nanoscale organization of endogenous proteins in neurons.
• Adaptability of the protocol for various adherent cell types.
• Successful visualization of protein distribution in both live and fixed cells.

Conclusions:

• SMLM is a powerful tool for nanoscale biological imaging.
• The provided protocol enables detailed study of endogenous protein organization.
• The methods are versatile for diverse cell types and imaging conditions.