Real-Time Microendoscopic Calcium Imaging in a Freely Moving Mouse

Begin with an anesthetized mouse secured in a mobile cage system.

The mouse has a pre-implanted gradient refractive index lens targeting the brain’s amygdala, where neurons express a calcium-sensitive fluorescent protein.

The lens surface is covered for protection.

Allow the mouse to awaken, then apply airflow for frictionless cage rotation.

Remove the lens cover and position a miniaturized microscope above the brain.

Turn on the microscope’s LED. Then, align the microscope so the lens is centered in the field of view.

Capture the lens image and adjust the microscope position.

As the cage rotates, the mouse’s leg movements generate fear, stimulating amygdala neurons.

This triggers an influx of calcium ions, which bind to fluorescent indicators.

The LED light excites these calcium-bound indicators, causing them to fluoresce.

The gradient refractive index lens efficiently captures and transmits this fluorescence to the microscope.

This enables real-time calcium imaging of amygdala neuronal activity.

To evaluate the quality of the GRIN lens implantation, place a carbon cage under the anesthetized mouse on the head bar of a mobile home cage system, and turn on the air flow so that the cage moves freely without friction. Use a microscope gripper and a stereotaxic manipulator to vertically align a miniaturized microscope to the frame, before lowering the microscope until the surface of the implanted GRIN lens appears in the image field of view within the software. Align the center of the implanted GRIN lens with the center of the field of view, and capture the image of the implanted GRIN lens surface. Then, slowly raise the objective lens of the microscope while observing the image for the appearance of GCaMP expressing cells.