Fig. 5: Electron microscopy images showing that aged intracellular condensates accumulate protein aggregates and lose phase-separated barrier.

a Correlative light electron microscopy (CLEM) of E. coli strain expressing synthetic condensates. A representative confocal image overview is shown (upper left), with the correlated region further visualized by EM (bottom left). Regions highlighted in yellow and orange boxes are magnified in the right panels. Cells were fixed using high-pressure freezing and embedded in resins. Thin-sections (100 nm) were incubated with an anti-GFP primary antibody, followed by a fluorescently tagged secondary antibody. Confocal microscopy images were aligned with TEM images based on cell structures and distribution. Scale bars, 10 μm (confocal) and 4 μm (TEM). b Representative tomographic slice images overview showing the ultrastructure of E. coli cells containing intracellular condensates at different stages (top). High-magnification of electron tomographic images reveal detailed condensate structures, with red arrows indicating protein aggregates (bottom). E. coli cells expressing intracellular condensates were fixed and examined using electron tomography. 250 nm thick TEM sections were imaged from multiple angels (± 60°) and aligned together using etomo software. Scale bars, 500 nm. c Three-dimensional (3D) models generated from tomographic reconstructions (top). Colored boxes indicate condensates at various stages and magnifications (bottom): (i), early-stage condensates (t_i = 2 h); (ii–iv), middle-stage condensates (t_i = 8 h) with protein aggregates; (v and vi), late-stage condensates (t_i = 14 h) showing protein aggregates and cytosolic infiltration. Cell membranes are highlighted in red, protein aggregates in white (middle-stage condensates) and blue (late-stage condensates). The colored surfaces around the condensates represent pseudo-barriers between condensates and cytoplasm. All experiments have been repeated at least three times with consistent results.