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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.
Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview01:13

Attenuated Total Reflectance (ATR) Infrared Spectroscopy: Overview

Attenuated total reflectance (ATR) infrared spectroscopy is a powerful analytical technique used to study the composition of materials. It is widely employed in chemistry, materials science, forensic science, and other fields where sample characterization is required. ATR has several advantages over traditional transmission IR spectroscopy, including the requirement of little to no sample preparation and the ability to analyze a wide range of samples.
The ATR process begins by directing a beam...
Three-Dimensional Microscopy in Microbiology01:28

Three-Dimensional Microscopy in Microbiology

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...
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...
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.

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Related Experiment Video

Updated: Jun 14, 2026

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

Total internal reflection microscopy: a surface inspection technique.

P A Temple

    Applied Optics
    |March 25, 2010
    PubMed
    Summary
    This summary is machine-generated.

    Total Internal Reflection Microscopy (TIRM) reveals surface and near-surface structures in transparent samples. This advanced optical microscopy technique provides detailed insights into scattering sites, complementing existing methods for material and biological studies.

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    Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy
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    Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

    Published on: January 21, 2019

    Area of Science:

    • Optics
    • Materials Science
    • Microscopy

    Background:

    • Observing surface and near-surface structures in transparent materials is crucial for quality control and research.
    • Existing microscopy techniques like dark-field and phase contrast (Nomarski) microscopy have limitations in resolving certain surface details.

    Purpose of the Study:

    • To introduce and demonstrate Total Internal Reflection Microscopy (TIRM) as a novel technique for surface structure analysis.
    • To highlight TIRM's ability to provide additional information on scattering site size and depth compared to conventional methods.

    Main Methods:

    • Illuminating transparent samples from within using a well-collimated polarized laser beam at or above the critical angle.
    • Examining the air side of the surface with an optical microscope.
    • Developing two TIRM microscope configurations, including an attachment for a commercial Nomarski microscope.

    Main Results:

    • TIRM successfully visualized structure at and near the surface of transparent samples and thin films.
    • The technique provided enhanced information on the size and depth of scattering sites.
    • Demonstrated applications in studying surface polishing, cleaning, laser damage, ion milling, and thin film inclusions.

    Conclusions:

    • Total Internal Reflection Microscopy (TIRM) is a powerful and complementary technique for detailed surface and near-surface analysis.
    • TIRM offers unique advantages for studying scattering sites and has potential applications in both materials science and biology.