A ratiometric two-photon probe for Ca2+ in live tissues and its application to spinal cord injury model.

Ratiometric imaging with a small-molecule probe is important for the in-situ quantitative analysis of chemical events. We developed a ratiometric two-photon fluorescent probe (SCa1-IREF) derived from dual dyes with different Stokes shifts. This probe has two identical windows: a Ca2+-sensing window and an internal reference window, with eliminated FRET interference. SCa1-IREF shows a marked change in the ratio upon response with Ca2+, significant two-photon brightness, considerable selectivity for Ca2+, and cell loading ability with low cytotoxicity. The ratiometric two-photon microscopy images revealed that this probe could directly and quantitatively estimate Ca2+ in live neurons and various tissues including rat spinal cord tissue. The studies of spinal cord injury model revealed that the Ca2+ level was significantly affected by elapsed time after injury. These results will provide useful applications for in-situ [Ca2+]i imaging and for the development of effective ratiometric probes.