Luminescent single and dual sensors for in vivo imaging of pH and pO2 (2011)

This thesis describes the development and characterization of novel optical sensor materials for clinical in vivo applications. At first, biocompatible single- and dual sensors for the clinically important parameters pH and pO2 were realized using a time-resolved read-out. For the first time, spatial pH and/or pO2 distributions in cutaneous and chronic wounds of humans were visualized. Further, a novel read-out technique for Fluorescence Ratiometric Imaging (FRIM) is presented. Planar sensor membranes for photographing of oxygen-, pH-, or CO2 distributions were designed. Finally, a biocompatible in vivo sensor for simultaneous and continuous read-out of pH/pO2 based on the new technique is demonstrated. Chapter 1 gives a short introduction on the importance of pH and pO2 especially in dermatology. Further, a brief overview on measurement techniques for oxygen and pH, and optical sensor techniques is presented. Chapter 2 describes the instruments that are applied in this thesis. In chapter 3, a referenced luminescent sensor for 2D high-resolution imaging of pH in vivo is described. The time-resolved sensing scheme is based on time-domain luminescence imaging of FITC and using a ruthenium complex as reference. The biocompatibility of this sensor is warranted via a three step safety mechanism. First, the dyes were covalently bound to, or incorporated in microparticles. Second, the size of the microparticles hampers or at least slows down cellular uptake. Finally, the particles were immobilized in a hydrogel to prevent particle leakage. A set of in vitro experiments served to characterize the sensor. It covers the physiologically relevant range of pH 3 to pH 9. The biocompatibility of the sensor was tested by conducting experiments on cytotoxicity, cellular uptake, and singlet oxygen production. Several in vivo studies on human tissue pH comparing sensor and pH electrode signals served to validate the sensor. Next, spatial and temporal pH distributions during cutaneous wound healing were imaged via the sensor. The visualization of heterogenic pH distribution in a chronic ulcer demonstrates the potential of the sensor. An in vivo sensor to image physiological wound oxygenation is presented in chapter 4. The sensor is based on luminescence lifetime imaging of an oxygen sensitive palladium porphyrin. The dye was incorporated in a partially oxygen blocking polymer in order to extend the sensitivity matching the physiological range of 0 to 100 mmHg. Studies on split-skin harvest sites and the oxygen gradient of the stratum corneum were accomplished. The pH sensor particles of chapter 3 and the pO2 sensitive particles (chapter 4) were combined to form one dual sensor layer for simultaneous in vivo imaging of pH/pO2. The sensor is described in detail in chapter 5. The signals of the hybrid sensor were separated using different emission filter sets. The sensor served to visualize analyte distributions on split skin donor sites and chronic venous as well as vasculitc ulcers. Chapter 6 deals with a novel and straightforward concept for ratiometric imaging read-out referred to as RGB imaging or photographing. The technique relies on fluorescence ratiometric imaging and makes use of the fact that digital pictures are composed of three virtually independent (viz. a red, green, and blue) pictures that are separately stored in digital cameras. The camera serves as a rudimental "spectrometer" for wavelength separation. The imaging concept is illustrated with the help of a specially designed oxygen sensor membrane, and a sensor for intrinsically referenced imaging of CO2 with RGB read-out is described. Moreover, the proof of principle of a RGB pH sensor membrane is demonstrated. Finally, chapter 7 describes a referenced dual sensor for pO2/pH based on the RGB technique including its transfer from the lab bench to clinical application. The novel material uses all three color channels of the RGB camera. It comprises the utilization of an inert reference fluorophore in the blue channel, a pH dependent signal in the green channel, and an oxygen dependent signal in the red channel. The biocompatibility of the materials was studied and ensured. The sensor material was characterized in detail in vitro, and applied in vivo to simultaneously visualize pH and pO2 distributions in human tissue on intact skin and during cutaneous and chronic wound healing.