Fluorescence Lifetime Imaging of a Caspase-3 Apoptosis Reporter

Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used. Changes in FRET by fluorescence lifetime imaging microscopy (FLIM) were measured. Unlike FRET measurements based on fluorescence intensity, lifetime measurements are independent of reporter concentration and scattering of light in tissue, making FLIM a robust method for imaging in 3D environments. Apoptosis of breast cancer cells in 2D culture, spheroids, and in vivo murine breast tumor xenografts in response to a variety of genetic and pharmacologic methods implicated in apoptosis of cancer cells was studied. This approach for quantifying apoptosis of cancer cells is based on caspase-3 activity at single-cell resolution using FLIM. © 2017 by John Wiley & Sons, Inc.