Fluorescence-Based Monitoring of Mitochondrial Stress in Astrocytes

Begin with an astrocyte culture transduced to express a Mito-timer protein localized to their mitochondria.

Mito-timer acts as a mitochondrial biosensor, emitting green fluorescence in healthy mitochondria and transitioning to red fluorescence in stressed or damaged ones.

Microscopically select large and isolated astrocytes for imaging.

Using different excitation wavelengths, capture images for green and red fluorescence.

To analyze the images, select frames for both red and green channels, then choose merge channels.

Apply auto shading correction and run the image enhancement algorithm to enhance image clarity.

Select the desired morphological parameters of mitochondria for analysis.

Using the mean intensity tab, measure the mean intensities of red and green fluorescence.

Calculate the ratio between red and green intensities, which indicates the overall mitochondrial health of the astrocyte.