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Measuring the Lateral Diffusion of Plasma Membrane Receptors Using Raster Image Correlation Spectroscopy
Sara Makaremi1, Jose Moran-Mirabal2,3
1School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada.
Raster image correlation spectroscopy (RICS) quantifies live cell diffusion using confocal microscopy. This protocol details measuring lateral diffusion of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) in macrophage plasma membranes.
Area of Science:
- Cell biology
- Biophysics
- Microscopy techniques
Background:
- Live cell imaging requires precise methods to study molecular dynamics.
- Plasma membrane receptor diffusion is crucial for cellular signaling.
- Raster Image Correlation Spectroscopy (RICS) offers a powerful tool for such investigations.
Purpose of the Study:
- To provide a detailed protocol for RICS analysis.
- To measure the lateral diffusion of specific immunoreceptors in live cells.
- To demonstrate RICS application in macrophage models.
Main Methods:
- Utilizing standard commercial laser scanning confocal microscopes.
- Acquiring raster-scanned images of RAW 264.7 macrophage cell lines.
- Applying RICS analysis to extract diffusion coefficients.
Main Results:
- Successfully measured lateral diffusion coefficients for TLR2 and CD14.
- Demonstrated the feasibility of RICS for quantifying membrane receptor mobility.
- Provided a step-by-step guideline for RICS data acquisition and analysis.
Conclusions:
- RICS is an effective method for quantifying lateral diffusion of membrane proteins.
- The protocol facilitates the study of immunoreceptor dynamics in live cells.
- This method aids in understanding cellular signaling mechanisms.
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