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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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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

Updated: Mar 2, 2026

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy VA-TIRFM
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Near-Membrane Refractometry Using Supercritical Angle Fluorescence.

Maia Brunstein1, Lopamudra Roy2, Martin Oheim1

  • 1Brain Physiology Laboratory, CNRS UMR 8118, Paris, France; Fédération de Recherche en Neurosciences FR 3636, Faculté de Sciences Fondamentales et Biomédicales, Université Paris Descartes, PRES Sorbonne Paris Cité, Paris, France.

Biophysical Journal
|May 13, 2017
PubMed
Summary
This summary is machine-generated.

Accurately measuring cell refractive index (RI) is crucial for quantitative total internal reflection fluorescence (TIRF) microscopy. This study introduces a novel method using TIRF and supercritical angle fluorescence to directly determine RI in live cells.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Quantitative total internal reflection fluorescence (TIRF) microscopy is vital for live-cell imaging.
  • Accurate interpretation of TIRF data is hindered by an unknown cell refractive index (RI).

Purpose of the Study:

  • To develop and validate a method for directly measuring the average RI within the cell's evanescent field during TIRF imaging.
  • To enable more quantitative analysis of cellular dynamics using TIRF microscopy.

Main Methods:

  • Combines TIRF excitation with supercritical angle fluorescence emission detection.
  • Measures the critical angle from back-focal plane images to determine RI.
  • Requires only a removable Bertrand lens on a standard TIRF microscope.

Main Results:

  • Successfully measured local RI with subcellular resolution in various cell types (mouse embryonic fibroblasts, BON cells).
  • Demonstrated applicability across different cellular compartments (vesicles, plasma membrane, mitochondria, endoplasmic reticulum) and fluorescent labels.
  • Validated the method using diverse dyes and fluorescent-protein chimeras.

Conclusions:

  • This technique provides a direct and accurate method for determining cell RI, overcoming a key limitation in quantitative TIRF microscopy.
  • Enables improved imaging of axial vesicle dynamics, mitochondrial energy states, and identification of metabolically active cancer cells.