RH 414 is primarily used as a fluorescent probe in neuroscience and cell biology research. It is especially valuable for studying membrane dynamics and neuronal activity due to its ability to rapidly respond to changes in membrane potential. The compound is commonly applied in imaging studies to monitor real-time electrical activity in neurons and other excitable cells, providing insights into synaptic transmission and network behavior in brain tissue. Its fast response time and high sensitivity make it suitable for high-resolution optical recording in both in vitro and in vivo preparations. RH 414 is also used in combination with other dyes to perform multi-parametric imaging, allowing researchers to simultaneously track membrane potential changes and other cellular events.