Imaging intracellular protein dynamics by spinning disk confocal microscopy.

Area of Science:

• Cell Biology
• Microscopy
• Biotechnology

Background:

• The expanding repertoire of fluorescent proteins (FPs) has propelled live cell imaging into mainstream research.
• Observing intracellular dynamics requires high spatial and temporal resolution.

Purpose of the Study:

• To detail components of state-of-the-art spinning disk confocal (SDC) microscopy systems for live cell imaging.
• To provide guidelines for integrating SDC with other advanced microscopy techniques.
• To offer a protocol for generating stable cell lines expressing fluorescently tagged proteins.

Main Methods:

• Description of key components in an optimized SDC system.
• Guidelines for coupling SDC with total internal reflection microscopy and photoactivation.
• Protocol for lentivirus-mediated transduction for stable cell line generation.

Main Results:

• SDC microscopy achieves high-speed (up to 1000 fps) optical sectioning for live cell analysis.
• Specific design choices for SDC systems are explained.
• Integration strategies with other imaging modalities are outlined.

Conclusions:

• SDC microscopy is a powerful tool for studying intracellular FP dynamics.
• Optimized SDC systems and integration with other techniques enhance live cell imaging capabilities.
• Lentivirus-mediated transduction provides a method for creating stable fluorescently tagged cell lines.