| A great number of cellular processes can lead to local changes in proton concentration and temperature. So, it is desirable to be able to measure pH and temperature with non-invasive and spatially resolved methods. In this thesis, I describe two unique methods to measure pH and temperature using the environmental sensitivity of the proton transfer of fluorescent molecules. The first method is based on the detection of the temperature and pH dependent parameters associated with the blinking of fluorophores by fluorescence correlation spectroscopy (FCS). Employing EGFP as the probe, I used this method to characterize temperature increase at a laser focus due to light absorption in a thin liquid sample. Using pyranine as a probe, I extended the applicability of this method to a range of pH including physiological pH. Also, I investigated in details the effect of buffer composition on the blinking of the fluorophores. Then, I concluded that one limitation of this method is its strong dependence on buffer conditions, which are not well characterized in vivo. The second approach is a pH measurement method based on ratiometric imaging. This method is not as dependent on buffer condition. We improved on current ratiometric imaging techniques by demonstrating the possibility of using two-photon excitation. This method was used to measure pH in pyranine loaded vesicles created during receptor-mediated endocytosis of mu-opioid receptors expressed in HEK 293 cells. Preliminary results showed that the pH in the endocytic vesicles dropped to a value similar to those measurements in late endosomes roughly ∼ 10 minutes after triggering the endocytosis, and eventually, the pH reading reached a value similar to that of the pH of lysosomes. |
