Here we describe a two-photon microscope and laser ablation setup combined with optical tweezers. We tested the setup on the fission yeast Schizosaccharomyces pombe, a commonly used model organism. We show that long-term imaging can be achieved without significant photo-bleaching or damage of the sample. The setup can precisely ablate sub-micrometer structures, such as microtubules and mitotic spindles, inside living cells, which remain viable after the manipulation. Longer exposure times lead to ablation, while shorter exposures lead to photo-bleaching of the target structure. We used optical tweezers to trap intracellular particles and to displace the cell nucleus. Two-photon fluorescence imaging of the manipulated cell can be performed simultaneously with trapping. The combination of techniques described here may help to solve a variety of problems in cell biology, such as positioning of organelles and the forces exerted by the cytoskeleton.