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Keywords: Upconversion, Gap Junction, Imaging, Cells,Fluorescence, Tissue Phantom
Issue Date: 24-Jan-2011
Abstract: Non-Invasive imaging is an area that has always been of interest in life science applications. There have been methods developed to achieve this purpose and fluorescence imaging is one such that has been worked on. Nanoparticles with varied properties have been applied for biological imaging in general and also in fluorescence imaging. Upconversion Nanoparticles (UCNs) are materials that were developed for their electronic application but their optical properties have attracted the attention in the field of bioimaging. The advantage that have made these materials suited for bioimaging includes low auto fluorescence background or absorbance by the tissue due to their ability to be excited at Near Infra Red (NIR) wavelength, their fluorescence emission which can be tuned in the visible or NIR region with no blinking or photobleaching and application in deep tissue imaging with NIR excitation and NIR emission. Though these properties are reported based on the excitation wavelengths and emission of the nanoparticles, the depth of the penetration of the excitation light and fluorescence emission of these materials are still unknown. Further, applications demonstrated from perspective of imaging are focused mainly on cancer cell targeting and cancer imaging. Thus, an application was chosen to demonstrate the use of these particles with cells other than cancer cell lines. The imaging of gap junctions was chosen as in the recent times it has been reported to be important for transplantation of cells and also in cancer therapeutics. It would be an advantage to be able to do gap junction detection on live cells but the existing methods involve staining or dye transfer in fixed cells. Further the dye tends to bleach over time thus not allowing time based imaging. Hence, to be able to do live cell imaging and also to monitor the gap junctions over time the UCNs were proposed as an option. The synthesis methods and the surface modification of the nanoparticles allow use with biological systems. In this work two different routes of surface modification are demonstrated. One of the modification methods involves the use of an inert material like silica to obtain core/shell structure while the other technique uses ligands to form micellar structure which could allow multi-particle encapsulation. Both these materials allow conjugation of molecules on the surface to target specific receptors on the cells. The choice of the particles after modification was made based on the application needs. The application of interest was to image gap junctions formed between cardiac cells and cardiac cell/bone marrow stem cell in co-culture using these nanoparticles. This was chosen to demonstrate the use of these particles with sensitive cells and also to show the use of the non-photobleaching property to monitor changes over time. For this, the antibody specific to the gap junctions were conjugated to the surface and the samples tested on cardiac cells and the cardiac bone marrow stem cell co-culture with controls to test the targeting.Further, the depth to which the particles could be used for imaging was accessed using a solid tissue phantom prepared in the lab. The depth to which the gap junctions labeled on cells could be imaged was also assessed using these tissue phantoms. This study demonstrates that the visible UCNs can be used to image up to a depth of 1 mm and hence can be used to image vasculature that are 200-500 microns deep. With the NIR emitting UCNs an imaging depth of 5 mm could be achieved. With the NIR emitting UCNs which are targeted to gap junctions, the imaging depth was a few millimeters. This could allow the use of these particles to image deeper structures like cervical cancer by non-invasive means.
Appears in Collections:Ph.D Theses (Open)

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