Sub-micron Opto-Chemical Probes for Studying Living Neurons

摘要

We have fabricated sub-micron opto-chemical probes for pH, oxygen and calcium monitoring and demonstrated their application in intracellular and extracellular monitoring of neurons (cortical neuronal cultures and acute hippocampal slices). Using these probes, we have measured extracellular pH in the stratum radiatum of the CA1 region of mouse hippocampus upon stimulation of presynaptic Schaffer collateral axons. Synaptic transmission was monitored using standard electrophysiological techniques. We find that the local pH transiently changes in response to synaptic stimulation. In addition, the geometry of the functionalized region on the probe combined with high sensitivity imaging enables simultaneous monitoring of spatially adjacent but distinct compartments. As proof of concept we impaled cultured neurons with the probe measured calcium and pH inside as well as directly outside of neurons as we changed the pH and calcium concentration in the physiological solution in the perfusion chamber. As such these probes can be used to study the impact of the environment on both cellular and extra-cellular space. Additionally as the chemical properties of the surrounding medium can be controlled and monitored with high precision, these probes enable differential measurement of the target parameter referenced to a stable bath. This approach eliminates the uncertainties associated with non-chemical fluctuations in the fluorescent emission and result in a self-calibrated opto-chemical probe. We have also demonstrated multifunctional probes that are capable of measuring up to three parameters in the extracellular space in brain slices.