Investigation And Applications Of Fluorescence Microscopy Based On Surface Plasmonic Enhancement Effect

As the development of nanotechnology, materials and optical technique, luminescence nanomaterials are widely used in applications of quantum information technology, biolabeling, bioimaging, nanoscale sensing and so on. However, some luminescence nanomaterials cannot be performed perfectly for practical applications owing to the limitation of their own properties. In order to improve the optical properties of the luminescence nanomaterials for pratical applications, we investigated the fluorescence microscopy and optical properties of single photostable nanoparticles such as color centers in nanodiamond, doubling nanocrystals and lanthanide-doped upconversion nanomaterials based on surface plasmon enhancement effect induced by metal nanostructure, and discussed their applications in this dissertation. The following studies have been performed.1. The photophysical properties of single Nitrogen-Vacancy (NV) Centers in nanodiamonds were investigated as a single photon source. When coupling to a gold film of 50 nm, the photoluminescence of a single NV center in nanodiamond could be enhanced significantly with maintaining the optical properties as a single photon emitter. With the measurement and statistical analysis of the photophysical properties for a mount of individual single NV centers on the gold film and on the glass plate separately, we demonstrated that the single NV centers in the gold film showed lower saturation excitation power, higher saturation count rate, and shorter excited-state lifetime. The photoluminescence enhancement was due to the enhanced local field caused by surface plasmon resonance and the energy transfer between the surface plasmon and the excited states of the NV centers. The orientations of single NV centers on the gold film were also studied by defocused imaging and numerical simulations. Comparing with any other oriented single NV centers, the single NV centers with perpendicular orientation to the gold film showed higher fluorescence intensity and shorter fluorescence lifetime because of the higher coupling efficiency of local electronmagnetic field. These results would benefit the investigation of high efficient and long-term stable single photon source.2. The optical properties of the second harmonic generation (SHG) in sodium niobate (NaNbO3) nanocrystals were studied. We demonstrated the tip-enhanced SHG microscopy by scanning the laser-focused single NaNbO3 nanocrystal with a gold-coated tip. When the tip was at the vicinity of the single NaNbO3 nanocrystal, the SHG signal was significantly enhanced, and the enhancement factor was strongly dependent on the excitation polarization and power. When the excitation power reached 12 mW, an over 12-fold tip enhancement was observed. Taking the advantages of the photostability and no saturation effect in nonlinear process, the tip-enhanced SHG microscopy would a promising technique in applications of biolabeling and bio imaging.3. The upconversion properties of Yb3+/Er3+:NaYF4 nanoparticles were investigated. With the highly enhanced local electronmagnetic field induced by a gold-coated tip, we performed the tip-enhanced upconversion fluorescence microscopy of a single Yb3+/Er3+:NaYF4 nanoparticle and obtained a maximum enhancement factor of 11 as well as an improved spatial resolution. By investigating the optical properties of tip-enhanced upconversion emission from different transition separately, it indicated that the gold-coated tip influenced the upconversion process in Yb3+/Er3+:NaYF4 nanoparticles by improving the reception and the transimission of incident field, enlarged the absorption and emission cross-sections, and improved the upconversion efficiency.4. The application of UV upconversion fluorescence from lanthanide doped nanoparticles for biological applications was investigated. DNA lesion would be induced by UV irradiation, which would cause the blockage of the transcription and replication of the DNA and subsequently apoptosis of the cells. Because of that, a potential cell treatment was proposed. If Pr:YAG nanoparticles were inside the cancerous cells, the apoptosis of the cells would be trigged by the UV upconversion emission from Pr:YAG nanoparticles excited by the laser at 488 nm. With performing the cytotoxic test and intracellular uptake of nanoparticle, we demonstrated the apoptosis of the HeLa cells induced by UV upconversion, and excluded the suspect of the laser itself for death of the cells. Based on the experimental setup, UV dosage for the apoptosis of HeLa cells was also determined.