Quantitative structured illumination microscopy via a physical model-based background filtering algorithm reveals actin dynamics.

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.

Phase separation drives the self-assembly of mitochondrial nucleoids for transcriptional modulation.

Mitochondria, the only semiautonomous organelles in mammalian cells, possess a circular, double-stranded genome termed mitochondrial DNA (mtDNA). While nuclear genomic DNA compaction, chromatin compartmentalization and transcription are known to be regulated by phase separation, how the mitochondrial nucleoid, a highly compacted spherical suborganelle, is assembled and functions is unknown. Here we assembled mitochondrial nucleoids in vitro and show that mitochondrial transcription factor A (TFAM) undergoes phase separation with mtDNA to drive nucleoid self-assembly. Moreover, nucleoid droplet formation promotes recruitment of the transcription machinery via a special, co-phase separation that concentrates transcription initiation, elongation and termination factors, and retains substrates to facilitate mtDNA transcription. We propose a model of mitochondrial nucleoid self-assembly driven by phase separation, and a pattern of co-phase separation involved in mitochondrial transcriptional regulation, which orchestrates the roles of TFAM in both mitochondrial nucleoid organization and transcription.