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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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Three-dimensional imaging techniques are essential in cell biology, allowing researchers to visualize intricate cellular structures with high resolution. Two prominent methods, Differential Interference Contrast Microscopy (DIC) and Confocal Scanning Laser Microscopy (CSLM), provide distinct advantages for imaging live and thick specimens, respectively.Differential Interference Contrast MicroscopyDIC microscopy enhances contrast in transparent, unstained samples by converting phase...

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Related Experiment Video

Updated: May 31, 2026

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)
09:14

Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)

Published on: October 2, 2012

Whole-cell scan using automatic variable-angle and variable-illumination-depth pseudo-total internal reflection

Wei Sun1, Aoshuang Xu, Kyle Marchuk

  • 1Ames Laboratory, U.S. Department of Energy, Iowa State University, Ames, IA 50011, USA.

Journal of Laboratory Automation
|July 19, 2011
PubMed
Summary
This summary is machine-generated.

This study presents an enhanced total internal reflection fluorescence microscope (TIRFM) capable of precise z-position measurements of nanospheres. The modified microscope also enables full cell body imaging and controlled illumination depth in pseudo-TIRFM mode.

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Area of Science:

  • Microscopy and Imaging Technologies
  • Biophysics
  • Nanotechnology

Background:

  • Total internal reflection fluorescence microscopy (TIRFM) is crucial for high-resolution imaging near surfaces.
  • Existing TIRFM systems often lack precise control over illumination depth and sample positioning.
  • Accurate z-position determination of nanoscale objects is essential in cell biology and material science.

Purpose of the Study:

  • To develop and validate an automatic calibration and angle-scanning prism-type TIRFM system.
  • To enable precise z-position measurements of fluorescent nanospheres.
  • To achieve controlled illumination field depth and full cell body imaging in pseudo-TIRFM mode.

Main Methods:

  • Modification of a prism-type TIRFM for variable-angle operation (TIRFM and pseudo-TIRFM modes).
  • Implementation of a computer program for automatic calibration of laser illumination spot.
  • Measurement of fluorescence intensities at varying incident angles for z-position extraction.
  • Utilizing subcritical incident angles for pseudo-TIRFM imaging and vertical scanning.

Main Results:

  • Successful operation in both TIRFM and pseudo-TIRFM modes was achieved.
  • Accurate extraction of z-positions for fluorescent nanospheres near cell membranes was demonstrated.
  • Full cell body imaging via vertical scanning in pseudo-TIRFM mode was performed.
  • Controllable illumination field depth by adjusting incident angle or laser spot position was confirmed.

Conclusions:

  • The modified TIRFM offers enhanced capabilities for nanoscale measurements and imaging.
  • The system provides a versatile platform for studying biological samples at the cell membrane interface.
  • This advanced TIRFM facilitates precise localization and imaging of cellular structures and nanoparticles.