Near-Native Imaging of Label-Free Silver Nanoparticles-Triggered 3D Subcellular Ultrastructural Reorganization in Microalgae.

Understanding the spatial orientation of nanoparticles and the corresponding subcellular architecture events favors uncovering fundamental toxic mechanisms and predicting response pathways of organisms toward environmental stressors. Herein, we map the spatial location of label-free citrate-coated Ag nanoparticles (Cit-AgNPs) and the corresponding subcellular reorganization in microalgae by a noninvasive 3D imaging approach, cryo-soft X-ray tomography (cryo-SXT). Cryo-SXT near-natively displays the 3D maps of Cit-AgNPs presenting in rarely identified sites, namely, extracellular polymeric substances (EPS) and the cytoplasm. By comparative 3D morphological assay, we observe that Cit-AgNPs disrupt the cellular ultrastructural homeostasis, triggering a severe malformation of cytoplasmic organelles with energy-producing and stress-regulating functions. AgNPs exposure causes evident disruption of the chloroplast membrane, significant attenuation of the pyrenoid matrix and starch sheath, extreme swelling of starch granules and lipid droplets, and shrinkage of the nucleolus. In accompaniment, the number and volume occupancy of starch granules are significantly increased. Meanwhile, the spatial topology of starch granules extends from the chloroplast to the cytoplasm with a dispersed distribution. Linking the dynamics of the internal structure and the alteration of physiological properties, we derive a comprehensive cytotoxic and response pathway of microalgae exposed to AgNPs. This work provides a perspective for assessing the toxicity at subcellular scales to achieve label-free nanoparticle-caused ultrastructure remodeling of phytoplankton.