Reinhard, Björn M.Velasco, Leslie2025-03-272025-03-272025https://hdl.handle.net/2144/499652025Noble metal nanoparticles (NPs) have unique photophysical properties, including non-blinking and non-bleaching characteristics, making them highly compatible with smaller NP sizes. These features make them especially appealing for studying cellular dynamics, as their smaller size will not interfere with biological activity. When compared to bulk metal particles, excitation of the conduction band of nanoparticles by an external field transforms the behavior of the electrons into an oscillating behavior. This phenomenon is known as localized surface plasmon resonance (LSPR). Under optical excitation, the combined near-field enhancements lead to coupled interactions observed as spectral shifts in the red region. Interferometric scattering microscopy (iSCAT) is an interference-based imaging platform that has high sensitivity of weak scatterers and indefinite limit on observation time. While iSCAT alone is diffraction limited, combining it with metal nanoparticles allows us to break the diffraction limit, offering enhanced resolution and sensitivity. We developed a two-color iSCAT microscope with ratiometric detection to monitor size differences in Ag NPs. Here, we explain the selection of optical components for this home-built system and share insights gained from addapting iSCAT with a commercial upright microscope. We chose 405 and 445 nm laser diodes for the microscope since they are located at the higher and lower plasmon resonance of Ag. Furthermore, laser diodes are advantageous because they have shorter coherence lengths, which help minimize speckle patterns in the imaging field of view. We demonstrated how two-color iSCAT can distinguish differences in Ag nanoparticle size and monitor trends with respect to change in ambient refractive index. Additionally, we validated the sensitivity of two-color iSCAT as small as 5 nm in size, which may be useful as labels for future studies on EGFR structural conformations. Using two-color iSCAT, we were able to observe the assembly of PEG-tethered Ag NP dimers of varying lengths by streptavidin-biotin interactions. The spectral shifts observed on both wavelength channels allowed us to monitor the dimers formed by from dimers formed by 0.4 kDa and 3.4 kDa PEG tethers. To validate iSCAT observations, we imaged the monomer and dimer samples using scanning electron microscopy (SEM). We were able to correlate phenomena observed in iSCAT with the SEM by showing probability plots of formed dimers. In iSCAT, monomers (10 and 20 nm) appeared as dark contrast, with 10 nm dimers showing a larger intensity, and 20 nm dimers with bright contrast. The spectral shifts observed in both wavelength channels enabled us to study the assembly of small nanoparticle dimers. Cells have a complex background which makes them too difficult to study with a standard iSCAT microscope, this is what led to the addition of a third color to our microscope. In the final section of this dissertation, we describe the calibration of three-color iSCAT microscope by imaging Au and Ag NPs. While Ag has greater optical properties compared to Au, they are toxic and less stable for nano conjugation purposes. Therefore, we incorporated a 520 nm laser line, which is close to the peak plasmon resonance of gold. We demonstrated how three-color iSCAT can discern between Au and Ag NPs and how this can pave the way for a new biosensing tool to distinguish different cell receptors.en-USChemistryThesis/Dissertation2025-03-170009-0008-8252-7004