Imaging Intracellular ATP Dynamics Using FRET-Based Sensors in an Organotypic Mouse Hippocampal Slice

Secure an organotypic mouse hippocampal slice in an experimental chamber.

The slice contains transduced neurons and astrocytes expressing an ATP sensor with donor and acceptor fluorescent proteins.

Place the chamber on a fluorescence microscope stage and perfuse it with oxygenated ACSF.

Allow the slice to adapt to the buffered conditions.

Using transmission light, focus the slice and identify the area of interest.

Switch to fluorescence mode and excite the donor fluorescent protein to initiate fluorescence emission.

Upon ATP binding, the sensor undergoes conformational changes, enabling fluorescence-based energy transfer from the donor to the acceptor, which emits fluorescence at a longer wavelength.

Use a dichroic mirror and bandpass filters to isolate and separate donor and acceptor emissions.

Select a region without fluorescence for background subtraction. Then, identify target cells and record their fluorescence over time.

Calculate the FRET ratio to monitor intracellular ATP levels in real-time.

Prepare experimental ACSF and obtain a pH of 7.4 by bubbling it with 95% oxygen and 5% carbon dioxide through an inserted tubing connected to the carbogen supply for at least 30 minutes. Keep t