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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Related Experiment Video

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Imaging Plasma Membrane Deformations With pTIRFM
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Imaging plasma membrane deformations with pTIRFM.

Daniel R Passmore1, Tejeshwar C Rao1, Andrew R Peleman1

  • 1Department of Biological Sciences, Wayne State University.

Journal of Visualized Experiments : Jove
|April 22, 2014
PubMed
Summary
This summary is machine-generated.

This study introduces photo-truncated total internal reflection fluorescence microscopy (PTIRFM) to track membrane shape changes during exocytosis. PTIRFM reveals how proteins and lipids remodel membranes for vesicle fusion and content release.

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Area of Science:

  • Cell Biology
  • Biophysics
  • Membrane Dynamics

Background:

  • Exocytosis requires precise regulation of lipid bilayer shape changes during vesicle and plasma membrane fusion.
  • Membrane curvature is controlled by dynamic interactions between lipids and specialized proteins.
  • Understanding these membrane remodeling events is crucial for cellular function, but limited by analytical methods lacking sensitivity to curvature or temporal resolution.

Purpose of the Study:

  • To investigate the dynamics of exocytosis by focusing on lipid bilayer shape changes.
  • To develop and implement a novel analytical method, photo-truncated total internal reflection fluorescence microscopy (PTIRFM), to track rapid membrane remodeling during exocytosis.
  • To visualize and interpret submicron changes in chromaffin cell membrane orientation during dense core vesicle (DCV) fusion.

Main Methods:

  • Utilized PTIRFM to visualize rapid, submicron membrane orientation changes.
  • Employed a lipophilic carbocyanine dye (diD) to stain the chromaffin cell membrane, making it sensitive to evanescent field polarization.
  • Used sequential excitation with orthogonal laser polarizations (561 nm, p-pol, s-pol) and a 488 nm laser for vesicle visualization and fusion timing.
  • Triggered exocytosis using KCl solution and analyzed diD emission intensity changes to understand fusion pore dilation.

Main Results:

  • PTIRFM successfully visualized and interpreted rapid membrane shape changes during DCV fusion in chromaffin cells.
  • The method demonstrated sensitivity to membrane curvature and high temporal resolution, overcoming previous limitations.
  • Analysis of diD emission intensity changes provided insights into fusion pore dilation dynamics.

Conclusions:

  • PTIRFM is a powerful tool for studying membrane remodeling during exocytosis with high spatial and temporal resolution.
  • This technique enables a deeper understanding of the molecular mechanisms underlying membrane shape control in cellular processes like vesicle fusion.
  • The findings contribute to a roadmap of how membrane-shaping proteins and lipids function during exocytosis and content release.