Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution

Area of Science:

• Cellular and Molecular Biology
• Microscopy Techniques
• Biophysics

Background:

• Cellular dynamics impede precise imaging of molecular patterns using advanced microscopy.
• Chemical fixation, while stabilizing cells, can alter molecular states and is lethal.

Purpose of the Study:

• To develop a non-lethal method for imaging molecular patterns in living cells.
• To enable consecutive imaging of the same cell under dynamic conditions.
• To study ligand-induced receptor tyrosine kinase activation dynamics.

Main Methods:

• On-stage reversible cryo-arrest technique to immobilize living cells.
• Single-molecule localization microscopy (SMLM) for nanoscale imaging.
• Fluorescence lifetime imaging microscopy (FLIM) for microscale imaging.

Main Results:

• Demonstrated high cell survival rates and preserved growth factor signaling after reversible cryo-arrest.
• Successfully imaged nanoscale clustering of epidermal growth factor receptor (EGFR).
• Assessed microscale activity patterns of ephrin receptor A2 (EphA2) in endosomes.

Conclusions:

• Reversible cryo-arrest is a viable method for precise molecular pattern imaging in living cells.
• The technique preserves cellular dynamics and signaling pathways.
• Enables detailed investigation of molecular events at various scales without compromising cell viability.