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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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Related Experiment Video

Updated: May 9, 2026

Fluorescence Lifetime Macro Imager for Biomedical Applications
06:01

Fluorescence Lifetime Macro Imager for Biomedical Applications

Published on: April 7, 2023

Fluorescence lifetime imaging microscopy for quantitative biological imaging.

Leng-Chun Chen1, William R Lloyd, Ching-Wei Chang

  • 1Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA.

Methods in Cell Biology
|August 13, 2013
PubMed
Summary
This summary is machine-generated.

Fluorescence lifetime imaging microscopy (FLIM) offers high-resolution biological imaging by measuring fluorophore decay times. This technique enhances visualization and analysis of cellular structures and functions.

Keywords:
Fluorescence correlation spectroscopy FLIMFluorescence lifetime imaging microscopyFörster resonance energy transfer FLIMLaguerre polynomial fittingMultiexponential fittingMultiphoton FLIMMultispectral FLIMOptimal gatingPhasor analysisTotal variation denoising

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Area of Science:

  • Biophysics
  • Microscopy Techniques
  • Cell Biology

Background:

  • Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for cell biologists.
  • It provides microscopic spatial resolution for detecting, visualizing, and investigating biological systems.
  • Understanding fluorescence lifetime is crucial for its application in biological imaging.

Purpose of the Study:

  • To introduce the fundamental theory of fluorescence lifetime.
  • To discuss factors influencing fluorescence lifetimes and their advantages for image contrast.
  • To present experimental and data analysis methods for FLIM.

Main Methods:

  • Introduction to time- and frequency-domain experimental approaches for FLIM.
  • Discussion of data analysis techniques: rapid lifetime determination, multiexponential fitting, Laguerre polynomial fitting, and phasor plot analysis.
  • Methods for improving FLIM precision: optimal virtual gating and total variation denoising.

Main Results:

  • Detailed explanation of fluorescence decay characteristics and influencing factors.
  • Presentation of various FLIM data analysis methods for accurate lifetime determination.
  • Demonstration of techniques to enhance lifetime precision in FLIM maps.

Conclusions:

  • FLIM is a versatile technique for quantitative biological imaging.
  • Recent applications include Förster resonance energy transfer-FLIM, fluorescence correlation spectroscopy-FLIM, multispectral-FLIM, and multiphoton-FLIM.
  • FLIM significantly advances the study of structure and function in biological systems.