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 start perfusing it with a carboxygenated extracellular solution. Maintain a perfusion flow rate of 2 milliliters per minute and a temperature of 37 degrees Celsius in the recording chamber. Use a 20x, 0.95 NA water immersion objective and a CCD camera in combination with an infrared LED below the recording chamber to locate the retinal slice.

Start the two-photon imaging system, as indicated by the manufacturer. Next, turn on the laser and set it to 860 nanometers. Turn on the two detection channels for fluorescence imaging of ECFP and citrine. Next, using the image acquisition software, control the two-photon microscope to scan and select a row of cone terminals for recording. Set image acquisition to 128 by 16 pixel images or a similar configuration.

Restrict the scanned area to the cone terminals to avoid bleaching of photopigments in outer segments. Turn on the laser. Allow the cones to adapt to the scanning laser and the stimulus background light for 20 to 30 seconds before presenting light stimuli or applying pharmacological agents. Now, start the presentation of the arbitrary stimuli. The stimuli are generated by modulating the intensity of the two LEDs over time, using a microprocessor board controlled by customized software. Then, start recording the two fluorescence channels simultaneously using the respective image acquisition software.