in vivo quantitative FRET small animal imaging: intensity versus lifetime-based FRET

Area of Science:

• Biophysics
• Biomedical Imaging
• Optical Techniques

Background:

• Förster Resonance Energy Transfer (FRET) microscopy monitors molecular interactions and conformational changes (2-10 nm).
• FRET is expanding into in vivo optical imaging for drug-target engagement and release quantification in cancer models.
• Current methods include intensity-based FRET and macroscopic fluorescence lifetime (MFLI) FRET.

Approach:

• Compared intensity-based FRET (IVIS imager) and MFLI FRET for small animal optical in vivo imaging.
• Detailed analytical expressions and experimental protocols for quantifying FRET efficiency (E) and donor fraction (f) were established.
• Dynamic in vivo FRET quantified transferrin receptor-transferrin binding in mice, benchmarked against in vitro FRET.

Key Points:

• Both in vivo techniques showed similar dynamic trends for receptor-ligand engagement.
• MFLI FRET required only one measurement from a single mouse, compared to nine measurements from three mice for intensity-based FRET.
• MFLI FRET offers significant advantages in efficiency and animal usage.

Conclusions:

• MFLI FRET is the preferred method for longitudinal preclinical FRET studies, including targeted drug delivery in live mice.
• The study provides a detailed comparison of FRET quantification methodologies for in vivo applications.
• MFLI FRET enhances the feasibility of in vivo molecular interaction studies in preclinical settings.