A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Fluorescence Resonance Energy Transfer (FRET) sensors are crucial for studying cyclic Adenosine Mono Phosphate (cAMP), an important intracellular messenger.
• Existing Epac1-based FRET sensors utilize donor and acceptor fluorescent proteins (FPs) to detect changes in cAMP levels via conformational changes in Epac1.
• Detection methods commonly include Fluorescence Lifetime Imaging (FLIM) and Sensitized Emission (SE), often using ratio-imaging.

Purpose of the Study:

• To introduce and characterize a novel cAMP sensor, (T)Epac(VV), utilizing mTurquoise as the donor fluorescent protein.
• To evaluate the performance of (T)Epac(VV) for both FLIM and SE detection methods.
• To compare the new sensor's characteristics, including FRET span, signal-to-noise ratio, and photostability, against existing sensors.

Main Methods:

• Development of a new FRET-based cAMP sensor, (T)Epac(VV), incorporating mTurquoise as the donor FP.
• Assessment of sensor performance using Fluorescence Lifetime Imaging (FLIM) and Sensitized Emission (SE) techniques.
• Quantitative analysis of FRET span, signal-to-noise ratio, and photostability.

Main Results:

• The (T)Epac(VV) sensor demonstrates suitability for both FLIM and SE detection modalities.
• mTurquoise donor offers enhanced quantum efficiency and single-exponential fluorescence decay compared to CFP.
• The sensor exhibits outstanding FRET span, signal-to-noise ratio, and improved photostability.

Conclusions:

• The (T)Epac(VV) sensor represents a significant advancement in cAMP detection technology.
• Its dual applicability for FLIM and SE, coupled with superior performance metrics, makes it a versatile tool.
• (T)Epac(VV) is recommended as the preferred sensor for future cAMP research utilizing FLIM and ratiometric detection.