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In previous works, the dye exclusion method was used to examine cell death. Under this circumstance, the laser powers applied have to be high enough to compromise the cell membrane and induce instant cell death (necrosis). When a cell is labeled with gold nanoparticles, heat can be built up around the nanoparticles
instantly upon the irradiation of an ultrafast pulsed laser. If the laser power density is high enough, fragmentation of gold nanoparticles can be produced£¬which can bring a localized mechanical shock to the cell membrane causing membrane perforation. At a low power, when the energy fluence is high enough, the heat produced from gold nanorods can also burn the cell membrane. These two effects will compromise the integrity and cause leaking of cell membrane, leading to rapid cell death.As known, cell proliferation can be stopped either by damaging the cells (necrosis) or by inducing cell apoptosis. Necrosis is a process of cell death by
accident, which is normally due to the compromising of membrane integrity. In contrast, apoptosis is programmed cell death (suicide), which is a natural process that governs the proliferation of cells in a living body. As a matter of fact, what makes a cell cancerous is the disturbance of its natural apoptosis, leading to its proliferation out of control. To restore and enhance apoptosis, certain stimuli such as drugs and irradiation can be introduced. This forms the basis of the conventional chemotherapy and radiation therapy. With reduction in power density or energy fluence, the thermal effect becomes dominant. While keeping the membrane integrated, the photothermal effect of gold nanorods can potentially lead to the dysfunction of the subcellular structures that govern the proliferation of cells, causing cell apoptosis .While maintaining the therapeutic effects, a laser operating at these lower energy levels is clinically safer. In addition, compared with necrosis, apoptosis is more suitable to in vivo treatment since inflammation and even secondary cancers associated with necrosis can be avoided. Despite its significance, laser induced apoptosis of cancer cells in the presence of gold nanoparticles has not been investigated, to the best of our knowledge.
       In this work, gold nanorod-enhanced two-photon luminescence imaging and apoptosis of cancer cells will be investigated in the aim of developing a way for efficient and medically safe cancer detection and microsurgery using femtosecond lasers under twophoton microscopy. A two-photon Fluoview inverted scanning
microscope was used for two-photon excitation of gold nanorods. A human cervical cancer cell line HeLa was used as a model. To achieve efficient targeting of gold nanorods to the cancer cells, transferrin molecules were conjugated to the surface of the nanorods. Transferrin has been proven to be efficient in enhancing
cancer targeting by nanoparticles including gold nanoparticles.The imaging capability of gold nanorods was also compared with the cell autofluorescence and molecular dyes.

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