| Biophotonics is an exciting and fast-expanding frontier which involves a fusion of advanced photonics and biology. It has not only developed many novel methodologies for biomedical research, but also achieved significant results as an independent field. Aided with femtosecond (fs) laser technologies, important progresses have been made on manipulating, imaging, and engineering of biological samples from single molecules to tissues in the last 10 years. The laser beam of ultra-short pulses at near-infrared band enjoys a lot of advantages: high nonlinear efficiency, low absorption by biological samples, high spatial and temporal resolution with tight confinement, low photo-toxicity, non-invasive, and ease of control. In this thesis, we report new findings from cell manipulation by fs laser, including transfection, cell-cell fusion, and induction of apoptosis in cells, which are detailed as follows:;1. Transfection is a key technique in cell and molecular biology with many important biochemical applications. We selected a fiber fs laser at 1554 nm, an instrument widely used in optical communication research, as the excitation source. Our results demonstrated that the fs laser could perforate the cell membrane and the hole would close in sub-second interval after the laser exposure. We determined the safe exposure duration by detecting if there was any sign of mitochondrial depolarization at 1.5 hours after photoporation. Furthermore, we had successfully transfected HepG2 cells with a plasmid DNA containing the OFP gene, whose fluorescence could still be detected 24 hours after exposure. The transfection efficiency was as high as 77.3%. We also observed the proliferation of the transfected cells after 48 hours.;2. Cell-cell fusion is a powerful tool for the analysis of gene expression, chromosomal mapping, monoclonal antibody production, and cancer immunotherapy. One of the challenges of in vitro cell fusion is to improve the fusion efficiency without adding extra chemicals while maintaining the cells alive and healthy. We show here that targeted human cancer cells could be selected by an optical tweezer and fused by a finely focused fs laser beam at 1554 nm with a high fusion eftlciency. The result confirmed that human cells could be fused exclusively by fs laser pulses, and this is the first time human cells are fused together all-optically. Mixing of cytoplasm in the fused cells was subsequently observed, and cells from different cell lines were also fused. Based on these, we firstly developed the method of optical cell-cell fusion.;3. Failure in the induction of apoptosis or programmed cell death is one of the major contributions to the development of cancer and autoimmune diseases. Here we used a fs laser as a novel method to provide a direct apoptosis trigger to observe dynamic changes at subcellular level during apoptosis. First, we examined the effect of fs laser irradiation on the creation of reactive oxygen species (ROS) in exposed cells, which could trigger programmed cell death. By controlling the mitochondria electron transport chain (ETC), we investigated the mechanism of ROS generation by the fs pulses, including thermal effect and direct free electron liberation. Second, we induced apoptosis to targeted cells by the fs laser and found that the nuclear envelope (NE) formed tubular or tunnel-like structures (nuclear tubules - NT) inside the nucleus. The average number of NTs in each cell with laser treatment was significantly larger than in the control. Besides, the development of a NT was observed since its inception and it eventually merged with another one to form a larger NT. Meanwhile, mitochondria and tubulin were found inside the NT, and the NT formation always occurred after an upsurge of cellular Ca2+ concentration. More DNA fragmentation were also found in the region around the NTs. Based on this, we propose that NTs are developed during apoptosis and mitochondria migrate into the nucleus through the NTs to release death signals to trigger DNA fragmentation. Third, we used the fs laser to induce Ca2+ in cells in the form of a slow release, and firstly discovered that most Ca2+ was stored in the cytoplasm, and could diffuse into the nucleus after the optical trigger. Using fast confocal scanning, we obtained the path way of Ca2+ diffusion after the trigger in different cases. Our findings thus provide a new method of regulating the rate of apoptosis. |
