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Related Concept Videos

Total Internal Reflection Fluorescence Microscopy01:05

Total Internal Reflection Fluorescence Microscopy

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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Cell-substrate topology upon ALA-PDT using variable-angle total internal reflection fluorescence microscopy

Henri-Pierre Lassalle1, Harald Baumann, Wolfgang S L Strauss

  • 1Hochschule Aalen, Institut für Angewandte Forschung, Anton-Huber-Str. 21, 73430 Aalen, Germany.

Journal of Environmental Pathology, Toxicology and Oncology : Official Organ of the International Society for Environmental Toxicology and Cancer
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Summary

Total Internal Reflection Fluorescence Microscopy (TIRFM) revealed light-induced changes in human cancer cell proximity to substrates. Photosensitizer Protoporphyrin IX (PPIX) proximity decreased after light exposure, indicating cellular changes without detachment.

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Total Internal Reflection Fluorescence Microscopy (TIRFM) enables high-resolution imaging near cell membranes.
  • Evanescent fields in TIRFM allow precise control over imaging depth.
  • Photosensitizers like Protoporphyrin IX (PPIX) are relevant in photodynamic therapy.

Purpose of the Study:

  • To investigate the effect of light exposure on the cell-substrate distance in human cancer cells using TIRFM.
  • To analyze changes in Protoporphyrin IX (PPIX) localization and cell adhesion after light treatment.

Main Methods:

  • Utilized Total Internal Reflection Fluorescence Microscopy (TIRFM) with an electron multiplying (EM) CCD camera.
  • Administered 5-aminolevulinic acid (5-ALA) to induce PPIX fluorescence in human cancer cells.
  • Irradiated cells with sublethal light doses (635 nm, 4 J/cm2) and measured cell-substrate distances.

Main Results:

  • Cell-substrate distances, initially 20-250 nm (mean ~120 nm), decreased to below 100 nm after light exposure.
  • Focal adhesions, visualized using YFP-FAK, remained intact, suggesting no significant light-induced cell detachment.
  • PPIX fluorescence was studied in relation to these topological changes.

Conclusions:

  • Light exposure induces a measurable decrease in the cell-substrate distance in human cancer cells.
  • TIRFM is effective for quantifying nanometer-scale changes in cell-substrate interactions.
  • Cancer cells maintain focal adhesion integrity and substrate attachment despite light-induced proximity changes.