In previous works, we used the fluorescent dye 2-Me-4-OMe-TM as intracellular phosphate sensing through fluorescence lifetime imaging microscopy (FLIM). However, a lower sensitivity than expected was observed.
The use of fluorescent dyes needs the understanding of their photophysical properties for a correct interpretation of their signal. Because cytoplasm is a complex matrix with multiple compartments and macromolecules, we evaluated whether dye-macromolecule interactions or changes in the polarity of the microenvironment surrounding the dye could change the photophysical properties of 2-Me-4-OMe-TM.
We found that solvatochromism is the main reason of this anomalous behavior. Solvatochromism is the characteristic of a chromophore that undergoes a shift in its absorption and/or emission wavelengths when the substance is dissolved in solvents with different polarities. However, we have demonstrated that this effect can be used to detect differences in polarity between different regions of cells using FLIM microscopy. We found a specific pattern of intensity where the probe accumulates in some organelles. This finding allowed the isolation of these organelles to determine their polarity.
The manuscript has been published in the journal Dyes and Pigments. Congratulations to Laura Espinar-Barranco and the rest of co-authors.