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Related Concept Videos

Fluorescence and Phosphorescence: Instrumentation01:25

Fluorescence and Phosphorescence: Instrumentation

Fluorometers and spectrofluorometers are two types of instruments used for measuring molecular fluorescence. These instruments differ in how they select excitation and emission wavelengths and the type of light sources they utilize. Fluorometers use absorption interference filters to choose excitation and emission wavelengths. The excitation source in a fluorometer is typically a low-pressure mercury vapor lamp that emits intense lines distributed throughout the ultraviolet and visible regions.

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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
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Published on: January 18, 2017

A modified phasor approach for analyzing time-gated fluorescence lifetime images.

F Fereidouni1, A Esposito, G A Blab

  • 1Debye Institute, Utrecht University, Utrecht, the Netherlands. f.fereidouni@uu.nl

Journal of Microscopy
|September 22, 2011
PubMed
Summary

This study introduces a new phasor analysis method for time-gated fluorescence lifetime imaging. The approach enhances signal-to-noise ratio and retrieves complex lifetime data from biological specimens.

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Area of Science:

  • Biophotonics
  • Cellular imaging
  • Fluorescence spectroscopy

Background:

  • Fluorescence lifetime imaging (FLIM) maps cellular biochemical environments.
  • Time-correlated single photon counting (TCSPC) and time-gating are robust FLIM techniques.
  • Phasor analysis offers intuitive visualization and improved signal-to-noise ratio (S/N) in FLIM.

Purpose of the Study:

  • To develop a theoretical framework overcoming limitations of phasor analysis with time-gated FLIM data.
  • To improve the analysis of time-gated fluorescence lifetime imaging data.
  • To retrieve complex lifetime information from biological samples using time-gating.

Main Methods:

  • Proposed and validated a modified theoretical framework for phasor analysis.
  • Applied the method to simulated lifetime images and cellular samples.
  • Utilized global analysis principles adapted for time-gated data.

Main Results:

  • Successfully retrieved two distinct lifetimes from time-gated data, which standard methods could not resolve.
  • Demonstrated the method's ability to overcome under-sampling and decay curve truncation inherent in time-gating.
  • Showcased enhanced information retrieval from typical FLIM measurements.

Conclusions:

  • The developed phasor analysis framework effectively addresses challenges in time-gated FLIM.
  • This approach simplifies data analysis and increases the information obtainable from time-gated fluorescence lifetime imaging.
  • The method holds potential for more detailed biochemical mapping in cellular studies.