Development of Nonlinear Microscopy for Studies of Metabolism and Tissue Engineering

Licentiate thesis, 2009

Nonlinear microscopy techniques provide abilities for noninvasive and selective imaging of structural and chemical properties of biological systems. These techniques include coherent anti-Stokes Raman scattering (CARS) microscopy with the ability to selectively image molecular vibrations, second harmonic generation (SHG) microscopy which selectively images fiber structures, and third harmonic generation (THG) microscopy which is sensitive to interfaces between materials with different optical properties. These techniques can also be combined with fluorescent labeling and imaging by means of two-photon fluorescence (2PF) microscopy. In this thesis, I present four projects exploring development and applications of these techniques. In the first project, CARS microscopy was used together with 2PF, to establish a method to image lipid droplets in living yeast cells without labeling. The method was further used to quantitatively investigate differences in lipid storage depending on different nutritional and genetic background. Secondly, CARS microscopy was for the first time used to image glucose, not only in a simple glucose solution, but also in the surroundings of yeast cells, where the response from the cells was investigated spectrally. In the next project, a combination of SHG and CARS microscopy was used to follow the production of bacterial cellulose in order to produce artificial blood vessels, a project which may lead to optimized microstructures of the cellulose. Finally, CARS and THG microscopy were compared experimentally. This project gives new ideas on how to combine these exciting techniques in future studies, giving abilities of obtaining more visual information on biological systems in vivo.