Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (d(xy) approximate to 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (d(x-y) approximate to 110 nm) and axial (d(x-z) <= 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (d(x-y) approximate to 120 nm), 10 times greater than unstructured IRM (d(x-y) approximate to 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.