Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices
Transcription
Take a stimulation pipette containing red calcium-insensitive fluorophores in artificial cerebrospinal fluid, or ACSF.
Position the pipette near the target neuron in a hippocampal slice.
Place a patch pipette with a recording electrode bathed in an electrolyte containing red calcium-insensitive and green calcium indicator fluorophores near the target.
Apply constant positive pressure to briefly disturb the tissue surface, indicating proximity to the target.
As the dimple forms on the neuronal membrane, release the pressure to form a seal.
Apply negative pressure to tighten the seal.
Stabilize the membrane potential at a constant negative voltage.
Continue with negative pressure to break the membrane, allowing the fluorophore-containing electrolytes to spread within the neuron.
Visualize the red fluorescence of the target dendritic spine and align the stimulation pipette near it.
Apply electrical pulses to open the calcium channels.
Use two-photon microscopy to visualize transient changes in calcium levels as indicated by green fluorescence.
