Inscoper fastFLIM applications

The Inscoper fastFLIM, a camera-based time-domain fluorescence lifetime imaging microscopy (FLIM) system, is the fastest lifetime imaging technique designed for live cell experiments. This technology can be used :

• to follow molecular interaction and biochemical activities;
• to monitor in real-time the molecular conformational changes by Förster’s Resonance Energy Transfer (FRET);
• to characterize autofluorescence;
• to determine lifetime biosensors for ions concentration;
• to measure the dynamic pH changes with spatial correlation.

Here, this video highlights the way the Inscoper fastFLIM can monitor in real time the activation of protein using fluorescent biosensors relying on the principles of FRET-FLIM.

Aurora kinase A autophosphorylation

Aurora kinase A (AURKA) is a serine-threonine kinase implicated in a large panel of biological pathways that activate upon its autophosphorylation of Thr288. One of its functions is to control the stability and fidelity of the mitotic spindle and the overall efficiency of mitosis. Although well characterized at the structural level, consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. Characterization of the AURKA autophosphorylation is made possible using genetically-encoded FRET biosensors. 

Protocol to engineer FRET biosensors

In this video made by the Journal of Visualized Experiments, Dr. Bertolin describes a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis (Bertolin et al, 2020). First, possible donor/acceptor FRET pairs are presented, and the research team shows possible cloning and insertion methods of AURKA FRET biosensors in cells. Then, a step-by-step analysis is realized for rapid FRET measurements by FLIM using the Inscoper fastFLIM. The easy-to-use graphical interface allows customization of the acquisition sequence. 

The Inscoper fastFLIM provides biological researchers a versatile tool to monitor in real time lifetime changes on biosensors with a high spatiotemporal resolution.