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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.
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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 developed.
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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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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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Related Experiment Video

Updated: May 25, 2026

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis
10:43

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Published on: November 6, 2019

Highly confined surface imaging by solid immersion total internal reflection fluorescence microscopy.

Lin Wang1, Cvetelin Vasilev, Daniel P Canniffe

  • 1Department of Physics and Astronomy, The University of Sheffield, The Hicks Building, Hounsfield Road, Sheffield, S3 7RH, UK. l.wang@sheffield.ac.uk

Optics Express
|February 15, 2012
PubMed
Summary
This summary is machine-generated.

We developed solid immersion total internal reflection fluorescence (SITIRF) microscopy for highly confined surface imaging. This advanced technique significantly reduces imaging depth, enabling high-resolution, high-contrast visualization of biological samples.

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Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

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Area of Science:

  • Microscopy
  • Biophysics
  • Optical Engineering

Background:

  • Total internal reflection fluorescence (TIRF) microscopy is crucial for surface imaging.
  • Conventional TIRF microscopy has limitations in imaging depth and confinement.
  • Advancements in microscopy are needed for higher resolution and selectivity.

Purpose of the Study:

  • To introduce a novel solid immersion total internal reflection fluorescence (SITIRF) microscopy technique.
  • To explore the use of a high-refractive-index aplanatic solid immersion lens (ASIL) for enhanced TIRF imaging.
  • To demonstrate SITIRF microscopy's capability for highly confined surface imaging with reduced depth.

Main Methods:

  • Integration of a high-refractive-index aplanatic solid immersion lens (ASIL) into TIRF microscopy.
  • Utilization of zirconium dioxide, a material with high refractive index and low optical dispersion.
  • Development of a versatile system design for both epi-fluorescence and TIRF modes with variable illumination angles.

Main Results:

  • Achieved significantly reduced imaging depth, confined to tens of nanometers.
  • Demonstrated highly selective imaging capabilities using both synthetic and biological samples.
  • Obtained high-resolution and high-contrast imaging essential for detailed surface analysis.

Conclusions:

  • SITIRF microscopy offers superior surface imaging confinement and reduced depth compared to conventional TIRF.
  • The developed system provides flexibility in illumination modes and angles.
  • SITIRF microscopy holds significant potential for advanced biological imaging and related fields.