Two-Photon Microscopy for Monitoring Calcium Dynamics in Mouse Retinal Cone Photoreceptors

Place a coverslip containing membrane-mounted mouse retinal tissue under a two-photon microscope.

The tissue expresses a FRET-based calcium biosensor in cone photoreceptors.

Using a water immersion objective, focus on the tissue.

Under background illumination, cone axon terminals remain depolarized, allowing calcium influx.

Intracellular calcium binding alters the biosensor’s conformation, bringing the donor and acceptor fluorophores closer.

Begin two-photon imaging.

The microscope’s laser focuses low-energy near-infrared light into the tissue.

At the laser’s focal point, two photons simultaneously excite the biosensor, triggering energy transfer from the donor to the acceptor.

This increases the acceptor fluorescence while decreasing the donor fluorescence.

Calculate the fluorescence ratio.

Next, deliver light stimulation to hyperpolarize the cones, reducing intracellular calcium levels.

This reverts the biosensor's conformation, inhibiting energy transfer from the donor to the acceptor and lowering the fluorescence ratio.

Record the responses to analyze light-evoked calcium dynamics in cone axon terminals.

Transfer a slice from the holding chamber to the recording chamber and immediately sta