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The use of fluorescence has become commonplace in the biological sciences, with many studies utilizing probes based on commercially available fluorophores to provide insight into cell function and behavior. As these imaging applications become more advanced, it becomes increasingly important to acquire accurate quantitative measurements of the fluorescence signal. Absolute quantification of fluorescence, however, requires the fluorophores themselves to be insensitive to environmental factors such as nonspecific protein interactions and pH. Here, we present a method for characterizing the sensitivity of fluorophores to the cytosolic environment by comparing their fluorescent intensity to an environment-insensitive reference signal before and after intracellular delivery. Results indicated that although the fluorescent intensity of a few fluorophores, e.g., fluorescein, were highly susceptible to the intracellular environment, other fluorophores, e.g., Dylight 649, Alexa647, and Alexa750, were insensitive to the intracellular environment. It was also observed that the sensitivity of the fluorophore could be dependent on the biomolecule to which it was attached. In addition to assessing the environmental sensitivity of fluorophores, a method for quantifying the amount of fluorophores within living cells is also introduced. Overall, the present study provides a means to select fluorophores for studies that require an absolute quantification of fluorescence in the intracellular environment.

Assessing the Sensitivity of Commercially Available Fluorophores to the Intracellular Environment