<p>It is necessary to elucidate cellular internalization and intracellular biotransformation in order to accurately assess the toxicity and fate of nanoparticles after interaction with organisms. Therefore, this work employed a combination of high resolution imaging and <i>in situ</i> detection spectroscopic techniques to systematically investigate the intracellular localization, morphology and chemical speciation of silver in the cells of <i>Chlamydomonas reinhardtii</i>, a unicellular freshwater green alga, after exposure to AgNPs coated with polyvinylpyrrolidone at a concentration of 2.0 mg/L. High resolution secondary ion mass spectrometry and high-angle annular dark field scanning transmission electron microscopy together with energy dispersive spectroscopy and selected area electron diffraction collectively confirmed that after 48 h of exposure, AgNPs entered the periplasmic space after cellular internalization into the algal cells. Silver was also found to coexist with sulfur inside the cytoplasm in both crystalline and amorphous forms, which were further identified as β-Ag<sub>2</sub>S and silver thiolates with synchrotron X-ray absorption spectroscopy. In combination, these analyses demonstrated that silver inside algae could be attributed to the uptake and sequestration of Ag<sup>+</sup> ion released from AgNPs, which was further sequestrated into cellular compartments. This study provides solid evidence for particle internalization and biotransformation of AgNPs after interaction with algae.</p
