| This work focuses on methodology development for single molecule/particle optical approaches to studying biological systems. Single molecule/particle experiments, which probe the behavior of individual nanoscale objects within an ensemble, make it possible to measure the probability distribution for microscopic properties, which is difficult to obtain from bulk measurements unless the components in the system can be synchronized. Successful retrieval of this probability distribution depends on the number of useful photons extracted, which in turn requires high-sensitivity detection as well as robust data analysis. In the first part of this work (Chapter 2 to 4), a novel photon-by-photon procedure is introduced to analyze single particle trajectories. Based on the intrinsic photon statistics and a well-established statistical analysis protocol, photon-by-photon analysis offers a more efficient and quantitative characterization of the single particles, demonstrated for both diffusion-type and immobilized single particle measurements. The second part (Chapter 5,6) concerns developments and applications of nonlinear optical microscopy. For the first time, molecular chirality provides contrast for imaging using optically-active sum frequency generation microscopy. Then in a clinical imaging demonstration, two-photon excited fluorescence and second-harmonic generation are simultaneously used to specifically image melanoma and collagen fiber bundles in unstained human skin cancer tissues. Although the microscopy images in this work do not achieve single molecule sensitivity, they present a preliminary characterization of signal strength. The third part of this work (Chapter 7) discusses recent advances in single molecule study and suggests future work. These improvements in probe design, experimental techniques, and data analysis algorithms will help to advance our understanding of the molecular machineries in biological systems. |
