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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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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.
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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
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Related Experiment Video

Updated: Apr 18, 2026

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins
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Fast and Sensitive Interferon-γ Assay Using Supercritical Angle Fluorescence.

Christian M Winterflood1, Thomas Ruckstuhl1, Stefan Seeger1

  • 1Physikalisch-Chemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; E-Mails: c.winterflood@pci.uzh.ch (C.M.W.); t.ruckstuhl@pci.uzh.ch (T.R.).

Biosensors
|January 15, 2015
PubMed
Summary

We developed a rapid immunoassay for Interferon-γ (IFN-γ) that is highly sensitive and significantly faster than traditional methods like ELISA, offering a new tool for biological research.

Keywords:
interferon-gammaone-step immunoassaypolymer test tubesupercritical angle fluorescence

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Area of Science:

  • Biochemistry
  • Immunology
  • Assay Development

Background:

  • Interferon-γ (IFN-γ) is a crucial cytokine in immune responses.
  • Current methods for IFN-γ detection, such as ELISA, are time-consuming and labor-intensive.
  • A need exists for faster, more sensitive diagnostic tools.

Purpose of the Study:

  • To develop and characterize a novel, rapid immunoassay for Interferon-γ (IFN-γ).
  • To establish the assay's sensitivity, linear range, and speed.
  • To demonstrate its potential as a replacement for ELISA.

Main Methods:

  • A one-step, solid-phase sandwich immunoassay was developed.
  • The assay utilizes single-use polymer test tubes with supercritical angle fluorescence detection.
  • Measurements were performed in real-time using a compact fluorescence reader.

Main Results:

  • The immunoassay achieved a limit of detection of 1.9 pM (30 pg/mL).
  • A linear concentration range spanning three orders of magnitude was established.
  • The assay provided results in just 12 minutes.

Conclusions:

  • The novel immunoassay offers a highly sensitive and rapid method for quantifying Interferon-γ.
  • This 12-minute assay can potentially replace conventional ELISA methods.
  • The technology enables real-time binding measurements with high sensitivity.