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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.

Frederik Görlitz1, Douglas J Kelly2, Sean C Warren2

  • 1Photonics Group, Department of Physics, Imperial College London; f.gorlitz14@imperial.ac.uk.

Journal of Visualized Experiments : Jove
|February 13, 2017
PubMed
Summary
This summary is machine-generated.

We developed an open-source, automated instrument for high-content analysis using fluorescence lifetime imaging (FLIM) to study protein interactions via Förster resonance energy transfer (FRET). This system enables screening of cell signaling and identification of binding partners in multiwell plates.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Biotechnology

Background:

  • Protein interactions are crucial for cellular functions.
  • Förster resonance energy transfer (FRET) is a powerful technique for studying molecular interactions.
  • High-content screening of protein interactions in cells is essential for drug discovery and understanding cellular processes.

Purpose of the Study:

  • To present an open-source, automated high-content analysis instrument for fluorescence lifetime imaging (FLIM).
  • To enable assaying protein interactions using FRET in fixed or live cells within multiwell plates.
  • To facilitate screening of cell signaling and identification of protein binding partners.

Main Methods:

  • Automated multiwell plate fluorescence lifetime imaging (FLIM).
  • Utilizing Förster resonance energy transfer (FRET) for protein interaction and biosensor studies.
  • Open-source data acquisition software (µManager) and analysis tools (FLIMfit for OMERO).

Main Results:

  • Demonstrated functionality of the automated multiwell plate FLIM instrument.
  • Presented exemplar data on HIV Gag protein oligomerization.
  • Showcased a time-course study of a FRET biosensor in live cells.

Conclusions:

  • The developed instrument provides a versatile platform for high-content screening of protein interactions using FLIM-FRET.
  • Open-source software and hardware facilitate practical implementation and data analysis.
  • This approach is applicable to studying both intramolecular FRET biosensors and intermolecular protein interactions.