No abstract

The emergence of super-resolution (SR) fluorescence microscopy has rejuvenated the search for new cellular substructures. However, SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment. Thus, we developed SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional optical diffraction tomography (ODT) with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving the mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum, and lysosomes. Using dual-mode correlated live-cell imaging for a prolonged period of time, we observed novel subcellular structures named dark-vacuole bodies, the majority of which originate from densely populated perinuclear regions, and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane. This work demonstrates the unique capabilities of SR-FACT, which suggests its wide applicability in cell biology in general.

Despite the prevalence of superresolution (SR) microscopy, quantitative live-cell SR imaging that maintains the completeness of delicate structures and the linearity of fluorescence signals remains an uncharted territory. Structured illumination microscopy (SIM) is the ideal tool for live-cell SR imaging. However, it suffers from an out-of-focus background that leads to reconstruction artifacts. Previous post hoc background suppression methods are prone to human bias, fail at densely labeled structures, and are nonlinear. Here, we propose a physical model-based Background Filtering method for living cell SR imaging combined with the 2D-SIM reconstruction procedure (BF-SIM). BF-SIM helps preserve intricate and weak structures down to sub-70 nm resolution while maintaining signal linearity, which allows for the discovery of dynamic actin structures that, to the best of our knowledge, have not been previously monitored.

Despite its wide application in live-cell super-resolution (SR) imaging, structured illumination microscopy (SIM) suffers from aberrations caused by various sources. Although artefacts generated from inaccurate reconstruction parameter estimation and noise amplification can be minimized, aberrations due to the scattering of excitation light on samples have rarely been investigated. In this paper, by simulating multiple subcellular structure with the distinct refractive index from water, we study how different thicknesses of this subcellular structure scatter incident light on its optical path of SIM excitation. Because aberrant interference light aggravates with the increase in sample thickness, the reconstruction of the 2D-SIM SR image degraded with the change of focus along the axial axis. Therefore, this work may guide the future development of algorithms to suppress SIM artefacts caused by scattering in thick samples. This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)'.