FRET-Based Ca²⁺ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound

Area of Science:

• Cellular Biology
• Biophysics
• Microscopy

Background:

• Fluorescence resonance energy transfer (FRET)-based biosensors enable dynamic visualization of molecular events in live cells.
• Spectral separation of fluorophores is crucial for distinguishing signals in dual FRET imaging.
• Targeted stimulation and biosensor delivery are key challenges in advanced live cell imaging.

Purpose of the Study:

• To develop a novel FRET-based calcium (Ca2+) biosensor using EGFP and FusionRed (FRET-GFPRed).
• To establish high-frequency ultrasound (HFU) as a method for precise single-cell stimulation and biosensor delivery.
• To demonstrate dual FRET imaging of cell pairs using distinct FRET biosensors.

Main Methods:

• Development of a new FRET-based Ca2+ biosensor (FRET-GFPRed) utilizing EGFP and FusionRed.
• Application of high-frequency ultrasound (150 MHz) for targeted subcellular stimulation and intracellular delivery of biosensors.
• Dual FRET live cell imaging using FRET-GFPRed and a YFP/CFP-based biosensor (FRET-CFPYPet).

Main Results:

• The FRET-GFPRed biosensor was successfully developed and differentiated from existing biosensors using distinct filter settings.
• High-frequency ultrasound enabled precise, repeatable stimulation of single cells and intracellular delivery of biosensors.
• Dual FRET imaging of cell pairs was achieved through sequential HFU-mediated delivery of FRET-GFPRed and FRET-CFPYPet.
• The FRET-GFPRed biosensor showed a consistent 10-15% FRET response to both ionomycin and HFU stimulation.

Conclusions:

• The FRET-GFPRed biosensor offers a valuable tool for Ca2+ dynamics imaging in live cells.
• High-frequency ultrasound provides a novel approach for targeted single-cell stimulation and dual FRET imaging.
• This combined technique advances the capability for studying cell-cell interactions and signaling pathways with high spatial and temporal resolution.