This study demonstrates a method for imaging mitochondrial oxidation in genetically modified primary neurons using a fluorescent protein-based redox indicator. The approach allows for real-time monitoring of mitochondrial dynamics in response to NMDA receptor activation.
Take a coverslip with genetically modified primary neurons in an imaging chamber filled with an imaging buffer.
These cells express a fluorescent protein-based redox indicator in the mitochondrial matrix.
Position the chamber under a fluorescence microscope, identify the cells under visible light, then switch to fluorescence imaging.
Apply two excitation wavelengths to stimulate the indicator and measure fluorescence emission.
The fluorescence intensity ratio at these wavelengths reflects the mitochondrial oxidation state.
Introduce NMDA to activate cellular NMDA receptors, causing intracellular calcium influx.
The increase triggers mitochondrial calcium uptake, inducing ROS production and mitochondrial
oxidation.
The ROS oxidizes the indicator, altering its fluorescence and increasing the signal ratio.
Introduce oxidizing agents to oxidize the indicator fully, maximizing the signal ratio.
Remove the buffer, then introduce reducing agents to reduce the dye completely, minimizing the signal ratio.
Use the maximum and minimum signal ratios to calibrate and quantify NMDA-induced mitochondrial oxidation.
For live imaging, set the time lapse interval to 30 seconds and duration to 25 minutes. Then, mount the cells, place the chamber on the microscope, and focus the cells as demonstrated previously. Switch to scanning mode and use the 488 nanometer channel in the live view to focus and locate the cells for imaging. Optionally, to increase the number of recorded cells per run, use the multipoint function to image two to three fields of view per coverslip.
Start the time lapse acquisition and record five images as a two-minute baseline recording. Add 500 microliters of three times NMDA solution to the chamber to achieve the final concentration of 30 micromolar and record additional 20 images as a 10-minute NMDA response. Next, add 500 microliters of four times DA solution to the chamber and record six more images. Aspirate the buffer from the imaging chamber and replace it with 1 milliliter of DTT solution. After recording 10 more images, end the recording and save the image series.
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