Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Area of Science:

• Biophotonics
• Microscopy techniques
• Fluorescence spectroscopy

Background:

• Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is vital for biological measurements.
• FLIM accuracy is highly sensitive to experimental parameters.
• Standardized calibration is needed to minimize errors.

Purpose of the Study:

• To develop and validate a comprehensive calibration and characterization protocol for frequency-domain FLIM systems.
• To identify and mitigate sources of error affecting lifetime precision.
• To demonstrate the optimized system's capability for accurate measurements in biological samples.

Main Methods:

• Utilized standard fluorescent molecules and reference biological samples with single and multiple lifetime components.
• Performed rigorous system calibration and acquisition parameter optimization.
• Evaluated system performance using metrics of accuracy and reliability.

Main Results:

• Identified key experimental parameters influencing FLIM precision.
• Demonstrated significant improvement in fluorescence lifetime measurement accuracy and reliability post-optimization.
• Successfully visualized Förster Resonance Energy Transfer (FRET) in living CHO cells.

Conclusions:

• The proposed calibration and optimization protocol enhances FLIM accuracy and reliability.
• This method is suitable for samples with multiple lifetime components and adaptable to various frequency-domain FLIM systems.
• Optimized FLIM enables high-precision fluorescence lifetime measurements in biological applications.