Novel Concepts for Functional High Resolution Microscopy

Functional imaging of biological cells and organs is one of the key techniques that drives new discoveries in life science research and medicine. In this thesis novel concepts for functional imaging at high spatial and temporal resolution are presented. In the first part, an imaging modality named Triplet Lifetime Imaging is developed. The technique allows oxygen consumption within single cells to be monitored at sub-cellular resolution. Measurement of tissue and cell oxygenation is important in order to understand cell metabolism. The method exploits oxygen induced triplet lifetime changes. The technique is applied to a biological cell system, employing as reporter a cytosolic fusion protein of -galactosidase SNAP-tag labeled with TMR. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg8]-vasopressin (AVP) induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays mono-exponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and sub-cellular level. The method is further applied to various other biological systems, demonstrating its versatility and the usefulness of this novel imaging modality. In the second part of this thesis, a concept for optical spectroscopy named nonlinear correlation spectroscopy (NLCS) is developed. The method allows monitoring of diffusing and flowing nanoparticles made of nonlinear optical material. NLCS is a method related to fluorescence correlation spectroscopy (FCS), but instead of fluorescence intensity fluctuations, NLCS analyses coherent field fluctuations of the second and third harmonic light. In bulk material, the third harmonic contribution vanishes due to the destructive interference of the third harmonic light generated in front of and behind of the focal field (Guoy phase shift). On the other hand, nanoparticles with dimensions comparable or smaller than the focal volume can generate strong higher harmonic signals. Particles based on non-centrosymmetric non-linear materials such as KNbO3 have been found to show a strong second and third harmonic signal. The method and the theory are introduced and NLCS results for diffusing polystyrene spheres (PS) as well as KNbO3 particles are presented. These spectroscopic results open the door for future extension into imaging concepts.