Correction for photobleaching in dynamic fluorescence microscopy: application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery.

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally. Photobleaching rates up to 5.0 10(-3) s(-1) were measured when applying power densities up to 0.2 W.cm(-2). By applying the three-compartment model, the model drug uptake rate of 6.0 10(-3) s(-1) was measured independent of the applied laser power. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization.