The intent of this proposal is to develop and characterize a new class of nanoparticles designed to operate as ultra-bright markers for second harmonic imaging microscopy (SHIM) and potentially for other nonlinear microscopy techniques. In order to accomplish this goal the investigators have established two objectives; the first objective is to fabricate and characterize several different types of SHIM enhanced nanoparticles using both lithographic and chemical synthesis techniques to give maximum flexibility. The second is to actually demonstrate SHIM imaging and hyperthermia in a biological setting. In order to accomplish this aim, the investigators will use bifunctional crosslinkers to apply a protective coating to the particles, making them biologically stable and non-toxic, and also easy to conjugate with an antibody or ligand (such as folic acid) to promote selective binding of the particles to target cells and tissues. The investigators will demonstrate the biocompatibility of the nanoparticles as well as their potential to perform targeted therapy through hyperthermia.
This project, "Ultra-Efficient Plasmonic Nanoparticle Markers for Second Harmonic Imaging", concerned the development of a new class of metallic nanoparticles designed to operate as ultra-bright markers for second harmonic imaging microscopy and a related nonlinear optical microscopy, Two-Photon Excitation Fluorescence. The metallic nanoparticles can be used in medical imaging, in fundamental biological studies, and in the development of ultrasensitive chemical sensors. Prior work had demonstrated that metallic nanoparticles exhibit the surface plasmon effect which causes the dramatic absorption and emission of light at specific wavelengths. This effect is very sensitive to the shape and size of the particles and also to the local chemical environment of the particles. The surface plasmon effect occurs when light impinges on a metal nanoparticle, causing the free electrons in the metal to oscillate in one or more resonating modes which result in the confinement of a light wave at the surface of the metal particle. The particle shape and size dictates the wavelength of the travelling light wave due to resonance effects. Simple particle shapes include spheres, rods, and triangular prisms while the size at which surface plasmon effects are important is typically below 500 nm for light in and near the visible part of the spectrum. These light waves travel along the metal nanoparticle surfaces and can have extremely high intensities, especially at the edges of particles such as the tips of nanotriangles, the corners of nanocubes, and the ends of nanorods. The very high light intensities can be used to investigate the physics of optical processes useful for imaging and chemical sensing such as fluorescence. Since the surface plasmon effects are very sensitive to the local environment of the nanoparticle, they can be used as a probe of that environment which is often an organic layer often attached to the particles to make them biocompatible for use in a living system and the local liquid composition which is often water with added ionic species and proteins in the case of a living system. Thus, understanding surface plasmon effects is fundamental for developing advanced optical imaging techniques for biological systems and ultrasensitive chemical sensors. The work in this project therefore focused on understanding how electromagnetic radiation, i.e. light, is absorbed and emitted from metallic nanoparticles as the shape of the nanoparticles is varied along with the composition of attached organic nanolayers and the local chemical environment of the particles. We learned how to create arbitrary and stable patterns of fluorescence in three dimensions inside the gel may find applications in tissue engineering and other areas where it is important to conduct imaging in turbid media. Microscopy imaging inside a specimen can normally be performed only to shallow depth because scattering disrupts the phase front of the light so that a well-defined focus cannot form. It is possible to overcome this limitation with adaptive optics that pre-adjusts the phase of the light to compensate for the scattering distortion, and makes deep imaging possible. However, to image inside the bulk of a scaffold using adaptive optics, it is necessary to first construct "guiding stars" or well-defined imaging patterns approximately every 100 nanometers. We demonstrated that it is possible to use Two-Photon Excitation Fluorescence to activate azidocoumarin in a gelatin gel which resulted in the formation of stable patterns of fluorescence that can be imaged, i.e. a "guiding star" prototype. If this can be made a standard technique, it would have far-reaching implications in medical and biological applications, where imaging with subcellular resolution in thick living tissue will become possible. We are currently pursuing this in further work. In other work, our studies of fluorescent particles at different distances above gold and silicon surfaces may be applied to the design and fabrication of new dynamically tunable photonic devices and structures that could be used in chemical sensing, for example. When the surface was a conducting gold film and the fluorescent particles were separated from a gold film by a thin polymer hydrogel whose thickness could be changed by varying the pH of water in which the nanostructure was immersed, we found that we could use these pH-actuatable polymer films to tune the plasmon resonance of a gold nanostructure consisting of gold spheres separated from a gold surface by the polymer film. We found that we could measure very large (up to 100 nm) shifts in the wavelengths at which plasmonic resonances occurred which shows the potential impact of actuatable films on photonic devices in which optical signal processing might be modulated or actuated by changing environmental conditions such as pH. In summary, the work performed in this project will have significant benefits to society as it is geared toward achieving breakthroughs in the rapidly emerging areas of medical imaging, chemical sensing, and nanotechnology.
