Simultaneous recording of multiple cellular events by FRET

The function of many sensors for measuring intracellular events is based on Forster resonance energy transfer (FRET). Here we demonstrate for the first time the use of multiple ratiometric FRET sensors in parallel through spatial and spectral resolution. We monitored three calcium-dependent signaling events by a cytosolic sensor for calcium/calmodulin-dependent protein kinase II alpha, a membrane-bound sensor for protein kinase C, and a translocating FRET probe based on annexin A4. This multiparameter imaging approach gives insight into the precise timing of cellular events within one single cell, thereby providing a major advantage over single-parameter protocols. This type of imaging will likely be important for high content cell analysis and screening efforts in the future.