Ultra-high-speed fluorescence imaging

For several years, ultrasound contrast agents are being investigated for their therapeutic capacity. These gas-filled coated microbubbles have the ability to enhance local intravenous drug delivery. Microbubbles can be loaded with drugs by adding them to the bubble surface of by incorporating them into the core of the bubble. Upon microbubble oscillation and/or disruption, following ultrasound insonation, the drugs are released locally to achieve a therapeutic effect while avoiding systemic exposure. Furthermore, the interaction of oscillating microbubbles with the vascular endothelial layer causes an enhanced uptake of drugs by the surrounding tissue. To visualize the process of ultrasound-triggered drug delivery, the use of ultra-high-speed imaging is required to resolve the nanoseconds timescales involved. To obtain sufficient optical contrast and resolution to visualize the release of nanometer-sized particles, fluorescence microscopy has to be employed as well. Combining both techniques is challenging, due to the extremely short exposure time in which sufficient signal has to be collected from a limited amount of fluorescent material. In this thesis the techniques of ultra-high-speed fluorescence imaging is described. By combining laser-induced-fluorescence, employing a 5W continuous wave (CW) laser, and the Brandaris 128 ultra-high-speed imaging facility a system was developed that allows for the visualization of fluorescently labeled microbubbles at a nanoseconds timescale. This imaging techique is applied to study the mechanisms of ultrasound-triggered local drug delivery using microbubbles: the release from liposome-loaded microbubbles and the subsequent uptake by a vascular endothelial cell. Furthermore, fluorescence imaging is used to aid the research of microbubble dynamics, since the absolute size of the bubble can be determined at a higher precision. This leads to more accurate input data for theoretical models. Finally, two novel designs of drug-carrying, hard-shelled ultrasound contrast agents are characterized. The first design consists of a multimodal contrast agent for photoacoustic and ultrasound imaging. The second therapeutic agent can be employed as a two-step drug delivery system. Both microcapsule designs are characterized experimentally. Their response to laser light and ultrasound, respectively, is compared to new physical models describing the activation dynamics of these microcapsules, which were found to be in very good agreement.