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-I_REF) 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-I_REF 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.