Browsing by Subject "Two-photon excitation"
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Item Investigation of gold nanoparticle accumulation kinetics for effective cancer targeting(2010-08) Park, Jaesook; Tunnell, James W.; Dunn, Andrew K.; Sokolov, Konstantin; Roy, Krishnendu; Krishnan, SunilGold nanoparticles (GNP) have been widely used as optical imaging and photothermal therapy agents due to their biocompatibility, simplicity of conjugation chemistry, optical tunability and efficient light conversion to heat. A number of in vitro and in vivo studies have demonstrated that they can be used as effective thermal therapy and imaging contrast agents to treat and diagnose cancer. As clinical applications of GNPs for cancer imaging and therapy have gained interest, efforts for understanding their accumulation kinetics has become more important. Given the recent demonstration of intrinsic two-photon induced photoluminescence (TPIP) of gold nanoshells (GNSs) and gold nanorods (GNRs), TPIP imaging is an efficient tool for investigating the microscopic distribution of the GNPs at intra-organ level. The following work explores these GNPs’ physical and optical properties for effective use of GNPs in TPIP imaging and examines the feasibility of using intrinsic TPIP imaging to investigate GNP’s biodistribution in bulk tumors and thin tissue slices processed for standard histology. Our results showed that GNPs yield a strong TPIP signal, and we found that the direct luminescence-based contrast imaging of GNPs can image both GNPs and nuclei, cytoplasm or vasculature simultaneously. Also, we present the effect of GNP morphology on their distribution within organs. Collected images showed that GNPs had a heterogeneous distribution with higher accumulation at the tumor periphery. However, GNRs had deeper penetration into tumor than GNRs due to their shape and size. In addition, GNPs were observed in unique patterns close to vasculature. Finally, we introduce single- and multiple-dose administrations of GNPs as a way of increasing GNP accumulation in tumor. Our results show that multiple dosing can increase GNP accumulation in tumor 1.6 to 2 times more than single dosing. Histological analysis also demonstrated that there were no signs of acute toxicity in tumor, liver and spleen excised from the mice receiving 1 injection, 5 injections of GNPs and trehalose injection.Item Single-particle tracking and its application in biophysical phenotyping(2018-09-13) Liu, Yen-Liang; Yeh, Tim H. C.; Dunn, Andrew K.; Xie, Chong; Chen, Chun-LiangSingle-particle tracking (SPT) has advanced our knowledge of molecular and cellular biology since its introduction in the early 90s. Whereas important discoveries made by SPT have changed our view of the plasma membrane organization and motor protein dynamics, experimental studies of intracellular processes using SPT are rather scarce due to the lack of 3D tracking technique, trajectory analysis algorithm, and validation. This dissertation describes my work on the validation of a 3D tracking technique and the development of a trajectory analysis algorithm; the developed SPT technique and algorithm was applied to visualizing biomolecules trafficking in live cells, measuring mechanical properties of cells, monitoring DNA conformational changes, and differentiating metastatic cells from benign or less-invasive cancer cells. First, we provided an overview of current SPT technology and a detailed description of our 2-photon 3-dimensional single-particle tracking microscope termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) and improvements to TSUNAMI. Second, we applied the TSUNAMI to investigate epidermal growth factor receptor (EGFR) trafficking in single cancer cells or multicellular cancer spheroids at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 minutes and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, a trajectory analysis algorithm is developed with 81% segment classification accuracy in the simulated movement experiments. This algorithm accurately retrieves the dynamics of EGFR trafficking from the 3D trajectories in which EGFRs travel from the plasma membrane into the deep cytosol via active transport. Third, we present an advanced version of TSUNAMI microscopy with the capability of two-color dual-particle tracking, which enables us to monitor the conformational changes of nicked or gapped double-stranded DNA and to quantify the DNA bendability in free solution with no surface interference. In the end, we develop an SPT-based biophysical phenotyping assay named Transmembrane Receptor dynamics (TReD) to differentiate metastatic cancer cells from less invasive ones. While derailed transmembrane receptor trafficking has been seen as a hallmark of tumorigenesis and increased metastatic potential of cancers, the transmembrane receptor dynamics has never been used as a physical biomarker for cancer detection. We tested a series of breast cancer and prostate cancer cell lines to validate the TReD assay and discovered the positive correlation between EGFR diffusivities and metastatic potentials. Form the instrumentation of TSUNAMI microscope and the development of trajectory analysis algorithm to their applications to the quantifications of EGFR dynamics and DNA conformational changes, we believe that the systems and methods developed in this work will provide life scientists with a powerful toolset for the future of biological research.