Live-Cell Total Internal Reflection Fluorescence (TIRF) Microscopy to Investigate Protein Internalization Dynamics
Tejeshwar C Rao1, Tomasz J Nawara1, Alexa L Mattheyses2
1Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
Methods in Molecular Biology (Clifton, N.J.)
|February 11, 2022
Summary
Total internal reflection fluorescence (TIRF) microscopy visualizes cell surface dynamics with high contrast and minimal background. This method is ideal for studying events like receptor endocytosis near the plasma membrane.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Biophysics
Background:
- Apicobasal and planar cell polarity involve dynamic events at the plasma membrane.
- Visualizing these membrane events requires techniques that minimize background noise and out-of-focus light.
- Total Internal Reflection Fluorescence (TIRF) microscopy offers optical sectioning for enhanced visualization near the cell surface.
Purpose of the Study:
- To present a general experimental and analysis pipeline using TIRF microscopy.
- To demonstrate the application of TIRF for studying cell surface receptor endocytosis.
- To highlight the versatility of TIRF for observing various dynamic plasma membrane processes.
Main Methods:
- Utilizing Total Internal Reflection Fluorescence (TIRF) microscopy.
- Implementing an optical sectioning approach to focus on the sample-coverslip interface.
- Developing a general pipeline for TIRF microscopy experiments and data analysis.
Main Results:
- TIRF microscopy provides high-contrast images with limited background.
- It effectively visualizes and tracks dynamic events occurring near the plasma membrane.
- The presented pipeline is suitable for studying receptor endocytosis.
Conclusions:
- TIRF microscopy is an effective tool for visualizing dynamic cell surface events.
- The developed pipeline facilitates the study of receptor endocytosis.
- This approach can be adapted for diverse plasma membrane dynamics research.
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