Particle-based nanosensors have over the last decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors is challenging and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis, and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pKa to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.
- pH measurements
- Fluorescence microscopy