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 the saline bubbled during the entire experiment. Then, switch on the fluorescent light source of the monochromator.

Transfer the organotypic slice culture into the experimental chamber. Place a grid on top of the organotypic slice culture with the frame down, not touching the culture, and the threads up, touching the membrane. Place the chamber on the microscope stage and connect it to the perfusion system.

Switch on the peristaltic pump at a flow rate of 1.5 to 2.5 milliliters per minute. Make sure there is no leaking of the perfusion system. Using transmission light, bring the cultured slice into focus and identify the area where experiments shall be performed.

Before starting imaging experiments, wait at least 15 minutes to allow slices to adapt to the saline conditions. Then, switch on the camera and the imaging software. Excite the donor fluorescent protein at 435 nanometers. Set the exposure time to between 40 to 90 milliseconds.

Then, insert the dichroic mirror and the filters into the beam splitter unit. Split the fluorescence emission at 500 nanometers with an emission image splitter, and employ bandpass filters at 482 plus or minus 16 and 542 plus or minus 13.5 nanometers to further isolate donor and acceptor fluorescence.

Select a region of interest apparently devoid of cellular fluorescence for background subtraction. Circle single structures of labeled tissue in the image on the screen to create ROIs. Set the frequency of image acquisition in the overall recording time. For experiments longer than 30 minutes, an acquisition frequency of 0.2 to 0.5 Hertz is recommended to prevent phototoxicity. Subsequently, start the recording.