Visualizing Protein Kinase A Activity in a Mouse Using Two-Photon Fluorescence Lifetime Imaging Microscopy

Secure an anesthetized mouse with a cranial window over its motor cortex onto a treadmill.

The mouse expresses a Protein Kinase-A reporter in cortical neurons.

Position the treadmill under a two-photon FLIM objective.

Add a water droplet between the objective and the cranial window.

Allow the mouse to wake up and acclimate to the treadmill.

Using bright-field illumination, locate the imaging region. Close the microscope enclosure to block external light.

Set the imaging parameters, initiate treadmill rotation to induce locomotion, and begin imaging.

Enforced locomotion activates PKA, which phosphorylates the reporter, bringing its donor and acceptor fluorophores closer.

The microscope directs an infrared laser, exciting the donor to transfer energy to the acceptor, reducing the donor’s fluorescence lifetime.

When locomotion stops, PKA and the reporter return to their inactive states, decreasing energy transfer and increasing the donor’s fluorescence lifetime.

Analyze the images to compare cortical PKA levels during rest and locomotion.

At least two weeks after cranial window installation, set the two-photon excitation laser wavelength to 960 nanometers and confirm a lack of response to toe