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

プロトコル

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 

1. FRET-based ATP Imaging (Figure 1)

1. Before the experiment, prepare E-ACSF and bubble it with 95% O₂/5% CO₂ for at least 30 min to obtain a pH of 7.4. Switch on the fluorescent light source (Xenon lamp) of the monochromator (Figure 1). Start the perfusion just before taking out the slice from the incubator.
   NOTE: Keep the saline bubbled with 95% O₂ and 5% CO₂ during the entire experiment.
2. Transfer the slice into an experimental chamber that is constantly perfused with freshly carbogenated E-ACSF using a peristaltic pump (Figure 1). Then, fix the slice with a grid. Place the chamber onto the microscope stage and connect the perfusion system. Gas-proof laboratory tubing is recommended for perfusion.
   NOTE: Experiments can be performed at room temperature or near physiological temperature, depending on the experimental design. Check the stability and reliability of the perfusion flow to avoid changes of focus induced by movement of the tissue and/or changes in shear stress. Standard perfusion velocities for slice work, used by us and many other laboratories, are 1.5-2.5 mL/min.
3. Bring the cultured slice into focus using a transmission light. Identify the area where experiments shall be performed (for example: the CA1 region of the hippocampus). Before starting imaging experiments, wait at least 15 min to allow slices to adapt to the saline conditions. For the configuration of the experimental setup, see Figure 1.
4. Switch on the camera and the imaging software. Then, select the proper filter cube.
5. Excite the donor fluorescent protein (eCFP) at 435/17 nm (~435 nm). Set the exposure time between 40 to 90 ms.
   NOTE: Strong exposure of slices to the fluorescent light may result in phototoxic effects.
6. Excitation at 435 nm results in emission at both 475 nm (eCFP; donor) and 527 nm (Venus; acceptor). Split the fluorescence emission at 500 nm with an emission image splitter and employ bandpass filters at 483/32 and 542/27 to further isolate donor and acceptor fluorescence. Strong expression might result in saturation of the detectors. In this case, you might use a neutral density filter to reduce the intensity of excitation.
7. Select a region of interest (ROI) apparently devoid of cellular fluorescence for background subtraction. Then, create ROIs delineating cell bodies.
8. Set the frequency of image acquisition and the overall recording time. For long (>30 min) experiments, an acquisition frequency of 0.2-0.5 Hz is recommended to prevent phototoxicity.
9. Start the recording. It is recommended to record at least 5 min under baseline conditions to ensure the stability of the preparation.
   NOTE: Adjust the focus of the cell during the recording if needed.

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結果

Figure 1: Configuration of the FRET imaging setup. (A) Schematic illustration of the different components and their spatial arrangement required for the FRET imaging setup. The a...

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