Naturally occurring biological nanoparticles (BNPs) and synthetic nanoparticles have a significant role in a wide range of biomedical applications. For instance, direct detection of BNPs, such as viruses, can provide new methods of viral diagnostics while synthetic particles can be used as labels to indirectly detect biomarkers for drug discovery. Therefore, developing advanced tools for nanoparticle detection has gained popularity in biotechnological research. One of the most exciting recent developments in BNP detection has been single particle (or digital) counting of individual particles which offers unprecedented sensitivity levels. However, standard optical techniques face a significant challenge for nanoparticle detection, due the weak optical contrast of sub-wavelength particles. Interferometric microscopy, overcomes the limitations imposed by particle size which allows for visualizing unresolved (diffraction-limited) optical signatures of sub-wavelength particles. Single-particle interferometric reflectance imaging sensor (SP-IRIS), is a widefield microscopy platform, developed by our group over the last years. SP-IRIS uses interferometric enhancement and a layered substrate to increase the optical contrast for the target particles of interest. While this microscopy technique has shown remarkable sensitivity levels for numerous applications including detection of viral particles and nucleic acids, it has remained a specialty tool due to the utilization of z-scan measurements for extracting the optical signature of particles. The z-scan measurements that consist of multiple frames acquired at different focal positions impose two major drawbacks. The first is the requirement of repeatable and high resolution scanning optics and the second is the time and computational processing power required to analyze the image stacks. In this thesis we describe a novel imaging method termed `pixel-diversity‘ IRIS (PD-IRIS), which aims to provide a more practical detection method for nanoparticles by eliminating the need for acquiring z-stacks. PD-IRIS is built upon SP-IRIS, however it introduces a paradigm shift for encoding the necessary optical signature of target particles. PD-IRIS compresses the relevant optical information within a single image frame rather than an image stack. This is achieved by using camera sensors that simultaneously record multiple spectral or polarization channels. Therefore, a single image can record distinct spectral responses of target particles with respect to different excitation wavelengths (multi-spectral PD-IRIS) or the distinct scattering characteristics with respect to polarization (polarization PD-IRIS). This dissertation presents a rigorous study for both PD-IRIS modes and demonstrates the practical applications of nanoparticle detection with proof-of-concept measurements. / 2024-01-16T00:00:00Z