Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

Area of Science:

• Cell biology
• Biophysics
• Microscopy

Background:

• Understanding protein dynamics in the plasma membrane is crucial for receptor signaling and cellular functions.
• Existing methods like single particle tracking (SPT) and photo-activation localization microscopy (PALM) face challenges with high particle densities and low signal-to-noise ratios.
• Analyzing molecular dynamics of fluorescent particles with intermittent excitation is difficult.

Purpose of the Study:

• To develop and validate a novel analysis approach for quantifying single protein dynamics using photo-activated (PA) microscopy.
• To overcome limitations of existing methods in analyzing molecular dynamics at high particle densities and low signal-to-noise ratios.
• To apply the developed method to study protein diffusion in live cell plasma membranes.

Main Methods:

• Applied spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series.
• Simulated PA images of diffusing particles with varying photo-activation, blinking, and bleaching rates.
• Simulated data with high particle densities and mobile objects to test robustness.

Main Results:

• Successfully applied STICS analysis to PA microscopy data to quantify protein dynamics.
• Determined the diffusion coefficient of epidermal growth factor receptor (EGFR) in live COS-7 cells.
• Revealed differences in diffusion maps between wild-type and mutant Lck in activated T cells.

Conclusions:

• Developed a robust analysis approach for live cell PA microscopy data based on image correlation spectroscopy.
• The method accurately quantifies spatio-temporal dynamics of single proteins in the plasma membrane.
• This approach provides new insights into protein dynamics and receptor signaling in live cells.