To begin, open a multichannel Z stack image in Fiji, and open the desired biosensor analysis script file. Click Run in the script editor window to run the script. Then enter the requested information in the dialogue window that appears.
Select the channel numbers of the numerator and denominator for ratio calculation. For mitochondria targeted HyPer7, the numerator is the channel excited at 488 nanometers and the denominator is the channel excited at 405 nanometers. Select the channel number of the transmitted light image if present, or zero if there is none.
Then choose the desired background subtraction method. If the background is uniform, click select an image area to measure the background level in the image. Afterward, choose the desired noise subtraction method to reduce random variation in the detector readout.
Click Select an image area. Select the fixed value option to enter a previously measured noise level, which usually works well if the imaging conditions are kept constant. For accurate and consistent detection of mitochondria, select a thresholding algorithm such as Otsu or Maximum Entropy.
Use the same algorithm for all images in an experiment, but ensure that mitochondria are accurately recognized. Then select the number of regions of interest per cell. For example, if measuring mother bud differences, select two.
Select the output folder where measurements and ratio images will be saved. For background or noise correction, choose Select an image area. Follow the prompts to draw a background area using the rectangle ROI tool and click Okay.
While analyzing individual cells or subcellular regions, draw regions of interest based on the brightfield image. The ROI does not need to precisely match the cell outline as only thresholded mitochondria within the ROI will be measured. After creating each ROI, press T to add the selected ROI to the ROI Manager.
Check Show All in the ROI Manager to document the marked cells. Each added region will appear as a numbered item in the ROI Manager list. If analyzing more than one ROI per cell, mark the ROIs in the same order for each analyzed cell.
After all the desired ROIs have been added to the ROI manager, click Okay in the Mark Cells dialogue window. Next, select the measurement table format. In the Multi Measure dialogue window, check measure-all-slices and one-row-per-slice options to produce a table with a desired format.
Use the process multi ROI tables. rscript to process the tables created with the one-row-per-slice option. Do not check the append-results option.
Save the output files to the selected folder using the script. Now open the ratio Z stack image in Fiji to generate a colorized ratio image. Then open the colorize_ratio_image.
ijm script file and click Run in the script editor window. A dialogue window prompting to enter the requested information will appear. In the unmodulated option, all mitochondrial pixels appear at the same brightness.
Some images may appear noisy as dim and bright pixels contribute to the ratio image. Use the intensity modulated option to reduce the noise. In the minimum and maximum displayed values method, choose values near the average minimum and maximum values observed in an experiment.
To ensure consistency, acquire all images using the same imaging conditions and display all images using the same minimum and maximum values. Then select the projection mode to project the Z stack and show the entire mitochondrial population before colorization. For a maximum intensity projection, select maximum.
To create an average intensity projection, select average. Finally, select the folder to save the colorized images. If the unmodulated option is selected, choose a color scheme in the dialogue window that appears.
Utilize the fire or rainbow RGB lookup tables built into Fiji or any desired lookup table in ImageJ's LUT format. Use the script to save the output files to the selected folder. The oxidized reduced ratio of mitochondria tagged HyPer7 showed a dose dependent response to hydrogen peroxide concentration, which reached a plateau at one to two millimolar externally added hydrogen peroxide.
Differences in mitochondrial hydrogen peroxide were observed within the yeast cells. A statistically significant decrease in hydrogen peroxide biosensor readout was detected in mitochondria in the bud compared to the mother cell.
