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Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy
Published on: May 1, 2012
Cell membrane orientation visualized by polarized total internal reflection fluorescence
S E Sund1, J A Swanson, D Axelrod
1Biophysics Research Division and Department of Physics, University of Michigan, Ann Arbor, Michigan 48109 USA.
Biophysical Journal
|October 8, 1999
Summary
This study introduces a novel fluorescence microscopy method using polarized total internal reflection (TIR) illumination to visualize membrane orientation variations in living cells. The technique quantifies submicroscopic membrane curvature and dynamics, revealing previously hidden cellular structures.
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Cell membrane orientation changes are crucial for cellular processes like endocytosis and exocytosis.
- Visualizing these submicroscopic orientation changes has been challenging with conventional methods.
Purpose of the Study:
- To develop and validate a fluorescence microscopy technique for visualizing membrane orientation.
- To map microscopic and submicroscopic membrane curvature and dynamics in living cells.
Main Methods:
- Utilized polarized total internal reflection (TIR) illumination with a membrane probe (diI-C(18)-(3)).
- Analyzed the ratio of fluorescence excited by perpendicular and parallel polarized light to determine membrane orientation.
- Applied image deconvolution to enhance contrast and resolution of TIR fluorescence images.
Main Results:
- The method successfully visualized membrane orientation variations in model lipid bilayers, erythrocytes, and macrophages.
- Generated spatially-resolved, time-course maps of membrane orientations in macrophages.
- Quantified low-visibility membrane structures and dynamics.
Conclusions:
- The polarized TIR fluorescence microscopy technique offers a powerful tool for studying membrane dynamics.
- It enables the identification and quantification of subtle membrane features and activities.
- This method advances our understanding of cellular membrane behavior.
