How can I calculate the pH of internal organelles using a ratiometric pH method? Which formulas should I use after acquiring the fluorescent measurements? I have employed Lysosensor Blue-Yellow and Acridine Orange for my experiments.
Alistar Moy
You have to calculate the ratio of fluorescence emission intensities at 2 different excitation wavelengths for each organelle. Convert this ratio to pH values by using standard curves for the ratio of fluorescence emission intensities versus pH.
Ex. for Lysosensor Blue-Yellow, calculate the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm (F450/405) for each organelle. Then, convert these ratios to pH values by using standard curves for F450/405 versus pH.
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