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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.
Imaging Biological Samples with Optical Microscopy01:18

Imaging Biological Samples with Optical Microscopy

Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...
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.
Overview of Microscopy Techniques01:22

Overview of Microscopy Techniques

The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...
Phase Contrast and Differential Interference Contrast Microscopy01:26

Phase Contrast and Differential Interference Contrast Microscopy

Phase-Contrast Microscopes
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
Overview of Electron Microscopy01:25

Overview of Electron Microscopy

The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.

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Related Experiment Video

Updated: May 31, 2026

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip
14:09

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Published on: November 16, 2019

Total internal reflection STED microscopy.

Travis J Gould1, Jordan R Myers, Joerg Bewersdorf

  • 1Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut 06510, USA.

Optics Express
|July 13, 2011
PubMed
Summary
This summary is machine-generated.

We developed total internal reflection STED microscopy, achieving ~50 nm lateral resolution and ~70 nm axial sectioning near the cover slip. This advanced technique enhances imaging detail and reduces photodamage for live cell studies.

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Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
06:43

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

Published on: May 3, 2022

Area of Science:

  • Optics and Photonics
  • Biomedical Imaging
  • Super-resolution Microscopy

Background:

  • Stimulated emission depletion (STED) microscopy offers sub-100 nm lateral resolution.
  • Conventional STED microscopy has limitations in axial resolution compared to lateral resolution.
  • Far-field fluorescence microscopy techniques often struggle with precise axial sectioning.

Purpose of the Study:

  • To integrate total internal reflection (TIR) illumination with STED microscopy.
  • To enhance axial resolution in STED microscopy.
  • To improve live cell imaging by reducing photodamage and photobleaching.

Main Methods:

  • Implementation of total internal reflection (TIR) illumination into STED microscopy.
  • Characterization of the microscope's performance using fluorescent bead test samples.
  • Application to immuno-stained microtubule samples.

Main Results:

  • Achieved approximately 70 nm axial sectioning near the cover slip.
  • Maintained approximately 50 nm lateral resolution.
  • Demonstrated effective imaging of biological samples like microtubules.

Conclusions:

  • Total internal reflection STED microscopy significantly improves axial sectioning capabilities.
  • The technique offers potential for reduced photo-bleaching and photo-damage in live cell imaging.
  • This method advances super-resolution microscopy for detailed cellular visualization.