Forster resonance energy transfer-based total internal reflection fluorescence reader for apoptosis

Area of Science:

• Biophysics
• Cell Biology
• Biomedical Engineering

Background:

• Apoptosis detection is crucial for understanding cellular health and disease.
• Existing methods for monitoring apoptosis can be limited by background noise and sensitivity.
• Forster resonance energy transfer (FRET) offers a sensitive mechanism for detecting molecular interactions and conformational changes.

Purpose of the Study:

• To develop and validate a novel fluorescence reader for sensitive detection of apoptosis.
• To utilize FRET on living cell surfaces for real-time monitoring of caspase activity.
• To establish a platform for drug screening and in vitro diagnostics at the nanometer scale.

Main Methods:

• A fluorescence reader employing multiple total internal reflections (TIR) to illuminate cell surface samples with an evanescent field.
• Utilizing enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) linked by a DEVD peptide sequence.
• Measuring changes in ECFP fluorescence intensity and fluorescence lifetime to detect DEVD cleavage during apoptosis.

Main Results:

• The system successfully detected apoptosis by monitoring the disruption of FRET upon DEVD cleavage.
• An increase in ECFP fluorescence intensity and fluorescence lifetime correlated with apoptosis.
• Selective excitation of membrane-associated fluorophores eliminated intracellular and background luminescence, enhancing signal-to-noise ratio.

Conclusions:

• The developed fluorescence reader is a sensitive tool for detecting apoptosis on living cell surfaces.
• The FRET-based method provides a robust platform for drug screening and in vitro diagnostics.
• This technology enables nanometer-scale analysis with high specificity and minimal background interference.