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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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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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Related Experiment Video

Updated: Jul 24, 2025

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Setting up multicolour TIRF microscopy down to the single molecule level.

Chiara Schirripa Spagnolo1, Stefano Luin1,2

  • 1NEST Laboratory, Scuola Normale Superiore, Piazza San Silvestro 12, I-56127, Pisa, Italy.

Biomolecular Concepts
|July 10, 2023
PubMed
Summary
This summary is machine-generated.

Researchers developed a multi-channel Total Internal Reflection Fluorescence (TIRF) microscopy system for simultaneous multicolour single-molecule imaging. This advancement enhances sensitivity and provides guidelines for advanced live-cell membrane interaction studies.

Keywords:
cell membranemulti-channel TIRF microscopymulticolour fluorescence microscopyneurotrophic receptorssingle-molecule imaging

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Advanced microscopy is crucial for detailed biological mechanism investigation.
  • Total Internal Reflection Fluorescence (TIRF) microscopy excels at cell membrane visualization, primarily in single-colour applications.
  • Existing multicolour TIRF setups are limited, hindering simultaneous multi-channel studies.

Purpose of the Study:

  • To describe the implementation of a multi-channel TIRF microscopy system for simultaneous two-channel excitation and detection.
  • To address challenges in achieving simultaneous single-molecule detection across multiple channels.
  • To provide guidelines for optimizing fluorescent labelling strategies for multi-channel TIRF experiments.

Main Methods:

  • Modification of a commercial single-colour TIRF setup to a multi-channel system.
  • Rigorous optimization of camera settings and background minimization for enhanced sensitivity.
  • Strategies for fluorescent labelling, including probe selection, efficiency, and reaction orthogonality.

Main Results:

  • Successful implementation of a simultaneous two-channel excitation and detection TIRF microscopy system.
  • Demonstrated applications at high molecule density and detailed analysis of challenges for single-molecule level detection.
  • Identified key factors in fluorescent labelling that impact experimental outcomes.

Conclusions:

  • The developed multi-channel TIRF system significantly advances capabilities for simultaneous multicolour single-molecule imaging.
  • Optimized setup and labelling strategies are essential for achieving high sensitivity in multi-channel TIRF experiments.
  • This work offers valuable guidelines for researchers studying interaction mechanisms on living cell membranes using advanced TIRF microscopy.