“Light beam as a tool for activation, modification or destruction of selected organelles or even molecules in the interior of a living cell”, this notion sounds like a topic of science fiction. Nevertheless, today it is science, in some cases close to becoming a clinical routine. The objective of this project is to assert the competence and competitiveness in the edge technology of biological and medical applications of fs-lasers, and to develop new tools that allow us to measure and understand phenomena that we could not investigate before. The possibility to manipulate biological objects in non-intrusive and non-destructive, or to initiate reactions and changes in the biological system, open up a broad avenue of new and exciting applications for the future. Light can be used as a tool for manipulating or processing of bio-matter, and as information carrier for characterization or imaging. In the present proposal we wish combine both aspects in one facility, namely in a femtosecond laser microscope for intracellular nano-surgery and intracelluar time resolved functional imaging. Therefore, we designed and built two ultra-short pulse laser based optical microscopes to allow quantitative two-photon fluorescence microscopy and laser surgery within living cells at sub-micrometer scales. We developed a new, highly sensitive and accurate method to determine the two-photon absorption cross-section of fluorescent dyes that does not require knowledge of the detection efficiency or of the quantum efficiency of the dye being studied. This method will allow scientists working in the expanding field of multiphoton microscopy to move beyond qualitative measurements to quantitative studies. The cross-section was determined by measuring the saturation behavior, which requires accurate characterization of the spatial and temporal profile of the beam. To do this, we developed a new far-field beam-profiling tool based on a fiber optic tapered CMOS-camera and a nonparaxial beam propagation model. The laser nanosurgery system allows precise ablation inside cells and was used to investigate Theileria (an intracellular parasite) inside its host cell. Using different laser parameters, we successfully perforated the Theileria membrane without damaging the living host cell, allowing dye present in the host cell cytoplasm to enter the parasite. Studies to transfect Theileria with DNA are underway. In collaboration with a geologist, we also applied fs lasers to fluid inclusion analysis in minerals, overcoming key obstacles to research in this field and realizing our goal of establish a state of the art fs-laser user facility at the University of Bern for basic and interdisciplinary research.