A green lifetime biosensor for calcium that remains bright over its full dynamic range

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Fluorescent biosensors typically switch between bright and dim states, leading to signal-to-noise ratio (SNR) issues in the dim state.
• Precise, quantitative measurements are hindered by low SNR in conventional biosensors.

Purpose of the Study:

• To engineer a novel green fluorescent biosensor, G-Ca-FLITS, with consistent brightness and improved SNR.
• To assess G-Ca-FLITS performance for calcium imaging in various cellular compartments and organisms.

Main Methods:

• Development of a green-emitting calcium sensor (G-Ca-FLITS) through modification of an mTurquoise-based sensor.
• Utilized fluorescence lifetime imaging microscopy (FLIM) for calcium concentration measurements.
• Evaluated sensor performance in mammalian cells (HeLa) and in vivo in *Drosophila* brains.

Main Results:

• G-Ca-FLITS exhibits high brightness in both calcium-bound and -unbound states, comparable to GCaMP3 and jGCaMP7c.
• Calcium-induced fluorescence intensity change is minimal (~30%), ensuring a stable SNR.
• Demonstrated negligible pH sensitivity in the physiological range.
• Observed heterogeneous mitochondrial calcium transients in HeLa cells using FLIM.
• Assessed G-Ca-FLITS and its turquoise predecessor for two-photon FLIM in *Drosophila*.

Conclusions:

• G-Ca-FLITS offers a robust tool for quantitative calcium measurements, overcoming SNR limitations of traditional biosensors.
• The sensor's stability and performance make it suitable for diverse applications, including organelle imaging and in vivo studies.
• G-Ca-FLITS represents a significant advancement in calcium sensing technology.