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In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET.

Jason T Smith1, Nattawut Sinsuebphon1, Alena Rudkouskaya2

  • 1Center for Modeling, Simulation and Imaging in Medicine (CeMSIM), Rensselaer Polytechnic Institute, Troy, New York.

Biophysical Reports
|May 30, 2023
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Summary
This summary is machine-generated.

Macroscopic fluorescence lifetime (MFLI)-FRET offers significant advantages over intensity-based FRET for in vivo imaging. MFLI-FRET requires fewer measurements and animals, making it ideal for preclinical drug delivery studies.

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Area of Science:

  • Biophysics and Biomedical Imaging
  • Molecular Interactions and Dynamics

Background:

  • Förster resonance energy transfer (FRET) microscopy is crucial for monitoring molecular interactions and conformational changes (2-10 nm range).
  • FRET is increasingly applied in vivo for drug-target engagement and release quantification in cancer models using labeled probes.

Purpose of the Study:

  • To compare intensity-based FRET (sensitized emission) and macroscopic fluorescence lifetime (MFLI) FRET for small animal optical in vivo imaging.
  • To detail analytical expressions and experimental protocols for quantifying FRET efficiency (E) and the fraction of donor molecules involved (p).

Main Methods:

  • Compared sensitized emission FRET (three-cube approach, IVIS imager) with MFLI-FRET (custom time-gated intensified CCD system).
  • Quantified in vivo transferrin receptor-transferrin binding dynamics in live mice using a near-infrared labeled FRET pair.
  • Benchmarked in vivo results against in vitro FRET using hybridized oligonucleotides.

Main Results:

  • Both in vivo imaging techniques showed similar dynamic trends for receptor-ligand engagement.
  • MFLI-FRET required significantly fewer measurements (1 vs. 9) and animals (1 vs. 3) compared to intensity-based FRET.
  • MFLI-FRET demonstrated significant advantages in efficiency and reduced experimental complexity for in vivo quantification.

Conclusions:

  • MFLI-FRET is the preferred method for longitudinal preclinical FRET studies, including targeted drug delivery in live mice.
  • The study provides detailed protocols for quantifying FRET efficiency and donor fraction in vivo.