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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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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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Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability
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Quantifying Endothelial Transcytosis with Total Internal Reflection Fluorescence Microscopy (TIRF).

Erika Jang1, Siavash Ghaffari2, Warren L Lee3,4,5

  • 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|February 26, 2022
PubMed
Summary

This study introduces a new method using total internal reflection fluorescence (TIRF) microscopy to visualize and quantify endothelial transcytosis. This technique accurately measures the transport of molecules like low-density lipoprotein (LDL) across single cells in real time.

Keywords:
EndotheliumExocytosisLow-density LipoproteinTIRF microscopyTotal internal reflection fluorescenceTranscytosis

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy Techniques

Background:

  • Endothelial transcytosis is crucial for molecular transport but challenging to study.
  • Previous methods like electron microscopy and transwells have significant limitations.
  • These limitations include confounding factors and a largely descriptive nature.

Purpose of the Study:

  • To develop and present a novel protocol for visualizing and quantifying endothelial transcytosis.
  • To overcome the limitations of existing transcytosis research methods.
  • To enable real-time, single-cell analysis of transcytosis.

Main Methods:

  • Utilized total internal reflection fluorescence (TIRF) microscopy to image the basal membrane of endothelial cells.
  • Applied fluorescently tagged low-density lipoprotein (LDL) to the apical surface of endothelial cells.
  • Quantified exocytosis at the basal membrane in real time using TIRF microscopy and MATLAB analysis.

Main Results:

  • TIRF microscopy effectively visualizes and quantifies apical-to-basal transcytosis in endothelial cells.
  • The method bypasses limitations of previous techniques, such as paracellular leakage and low transfection efficiency.
  • Real-time, single-cell measurements reduce background noise and provide precise data.

Conclusions:

  • TIRF microscopy offers a powerful and accurate method for studying endothelial transcytosis.
  • This protocol allows for the real-time quantification of ligand transport across endothelial monolayers.
  • The approach is adaptable for studying the transcytosis of various ligands, including albumin and high-density lipoprotein.