Quantitative Optical Microscopy of Microscale Soft Matter Systems

Abstract

Biological and soft-matter systems contain a wide variety of nano- and microscale particles whose physical properties, such as size, internal organization, and refractive-index contrast as a proxy for internal concentration, influence their function. Yet, most of these structures are weakly scattering and lie below the diffraction limit, making them difficult to access with conventional optical microscopy. Many existing characterization techniques rely on ensemble averaging or diffusion-based sizing, masking particle-level heterogeneity, and limiting quantitative insight into nanoscale biology and soft condensed matter. These challenges are particularly crucial for biomolecular condensates, whose submicrometer sizes, low refractive-index contrast, and dynamic internal structure place them outside the reach of standard imaging and scattering approaches.

This thesis presents two complementary interferometric methods for label-free, single-particle characterization across the nano–microscale. The first, Dual-Angle Interferometric Scattering Microscopy (DAISY), combines forward- and backward-scattered light with single-particle tracking to extract optical size, polarizability, refractive-index contrast, and hydrodynamic mobility for individual nanoparticles. This dual-channel design enables media-independent sizing and morphological inference by comparing optical size with mobility-based measurements. The second method integrates off-axis holographic microscopy with quantitative scattering analysis to characterize thousands of weakly scattering biomolecular condensates at the submicrometer scale. By reconstructing the full complex optical field and fitting appropriate scattering models, this approach retrieves condensate size, refractive index, internal mass distribution, interfacial structure, and hydrodynamic mobility. Applied to condensates formed by proteins, the method reveals salt-dependent structural changes, distinct interfacial morphologies, and dynamic behavior inaccessible to ensemble or diffusion-based techniques.

Together, these developments establish a unified, label-free framework for probing the structure and dynamics of nanoparticles and biomolecular condensates at the single-particle level. By bridging optical and mechanical observables, this work expands the range of soft-matter systems that can be quantitatively characterized and provides new tools for exploring the physical principles underlying phase-separated biological assemblies.