With wavelengths of 405, 470, and 580nm, the 405nm is used as a reference channel to control the data to effectively remove motion noise, while the 470 and 580nm can excite two types off luorescent proteins (e.g., calcium-sensitive fluorescent proteins or neuro transmitter probes, etc.).which can be used to detect the changes of different ions (e.g., calcium ions) or neurotransmitterconcentrations (e.g., dopamine, epinephrine, etc.), respectively.The system can be used in in vivoanimals to detect the level of signaling changes associated with different neuronal cell populations.to reflect the coding, characteristics of difierent types of neurons in the same behavioral paradigm,and to validate connections between neurons or correlations between signaling pathways.