Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes.

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Measuring mechanical forces within living cells is a significant technical hurdle in biological research.
• Understanding cellular forces is crucial for various biological processes, including cell division, migration, and tissue development.

Purpose of the Study:

• To introduce and detail the development of hydrophobic mechanosensing fluorescent probes, termed Flippers.
• To provide a comprehensive guideline for utilizing Flipper probes with fluorescence lifetime imaging microscopy (FLIM) to measure cellular membrane biophysical parameters.

Main Methods:

• Development of Flipper probes, which are hydrophobic fluorescent molecules whose fluorescence lifetime is sensitive to lipid packing.
• Technical optimization of Flipper probes for effective cellular imaging.
• Characterization of Flipper probes in diverse biological and in vitro systems using fluorescence lifetime imaging microscopy.

Main Results:

• Flipper probes demonstrate a measurable change in fluorescence lifetime correlating with alterations in membrane tension.
• Successful application of Flipper probes in various biological contexts, including cells, tissues, and organs.
• Demonstrated ability of Flipper probes to report on long-range forces within biological systems.

Conclusions:

• Flipper probes represent a valuable tool for quantifying membrane tension and cellular forces.
• Fluorescence lifetime imaging microscopy with Flipper probes provides a robust method for assessing membrane biophysical parameters.
• This technology enables new avenues for studying mechanobiology at cellular, tissue, and organ levels.