Label-free bio-imaging and quantification of polluting carbonaceous particles to assess their toxicology

In 2015, it is estimated that worldwide 4.2 million people died prematurely because of the consequences of air pollution. Moreover, ambient pollution has been identified as the leading cause of the global disease burden. The core of atmospheric pollutant particles is represented by combustion-derived particles (CDPs), such as carbon black (CB) and black carbon (BC), which are produced during incomplete combustion processes of, for example, diesel fuel. Various studies indisputably demonstrated that exposure to CDPs is associated with a wide range of adverse health effects including, among others, cardiovascular and pulmonary diseases as well as lung cancer. Recent investigations have even promulgated this type of particles to be more harmful to the human health than other subcomponents of particulate air pollution.

Although great progress has been made in comprehending the interactions of CDPs with biological systems, improved methods to probe these particles in complex environments and to gain detailed information about their corresponding adverse impact, are still needed. Hence, this dissertation focused on the persisting demand of analytical techniques for the qualitative and quantitative determination of carbonaceous particles in biologically relevant samples. Consequently, two novel optical-based analytical methods were developed using white-light generation under femtosecond pulsed laser illumination and pump-probe imaging. Both techniques were evaluated in various complex biological environments, such as human lung fibroblasts and urine samples. These results clearly showed that both techniques have numerous advantages over existing technology as these are label-free, biocompatible and straightforward approaches that discriminate background signals from biological components and can be easily combined with various fluorochromes. Furthermore, it was shown that both techniques can be employed to measure black carbon particles in urine and that urinary loading can serve as an exposure matrix to CDP-based air pollution, reflecting the passage of black carbon particles from circulation into urine.

Additionally, advances were made in the field of displacement microscopy by making the quantification of large cell-induced deformations possible and by studying these displacements in a label-free manner using second harmonic generation (SHG) from collagen fibrils instead of the traditionally used fiducial markers. In this dissertation, this advanced method has been employed to elucidate the toxicological effects of carbonaceous particles, which proved that those particles can inhibit lung fibroblast-mediated matrix remodeling via an oxidant-dependent mechanism.     

During the past four years, great advances have been made but we are not there yet. First of all, in light of our patent application, an automatized, prototype set-up needs to be built to make high-throughput screening of biological samples possible. Secondly, additional toxicological studies should be performed to answer crucial questions about particle internalization and co-localization inside cellular organelles and to confirm the proposed oxidant-dependent pathway. Thirdly, epidemiological studies should be set-up to closely study the effect of carbon-based materials on human health and golden standards based on optical microscopy should be re-evaluated.  

In summary, the two optical-based analytical techniques and advanced displacement microscopy method described in this dissertation pushed the boundaries of toxicological and epidemiological studies to the next level and opened doors to many new opportunities in both fundamental and applied research. We believe that eventually these contributions will result in an improved understanding of the specific toxicology effects of CDPs and direct associations of those particles with adverse health effects. It might even lead to novel or additional indicators for more precise air quality strategies and regulatory proposals.