I’ve been tasked with determining if fluorescein photobleaches and I have access to a dual beam UV-vis spectrometer and a fluorometer.
From what I understand, photobleaching is the photochemical destruction of the fluorophore molecule, such that the molecule can no longer fluoresce. In other words, photobleaching is the process in which the total number of ground state molecules is depleted through photochemical destruction when molecules are either in the singlet or the triplet excited state. From this, I gather than photobleaching will result in the molecule being unable to absorb photons and transition from the ground to excited state. Is it correct to assume fluorecein will no longer absorb at the desired wavelength after photobleaching?
Assuming I’m correct, I figure I can use UV-vis spectroscopy to measure the absorbance of fluorecein as a function of time. Then, assuming a first (or pseudo-first) order rate equation, the data can be linearized and the slope will correspond to the (pseudo-)first order rate coefficient, effectively providing me with information regarding the kinetics of fluorecien photobleaching.
Additionally, I will be investigating the effect that different factors, such as pH or presence of oxygen, has on the photobleaching of fluorecein.
I’m wondering if this is generally correct and where the procedure could be improved. Furthermore, I do not have any experience with a fluorometer, so if there is a better way to go about this using the fluorometer, I would greatly appreciate it if someone could let me know.