Label-Free Imaging of Humic Substance Bioaccumulation by Pump-Probe Microscopy.

Humic substances (HS) are the most abundant forms of natural organic matter on the earth surface. Comprised of decomposed plant and animal materials rich in carbon, oxygen, hydrogen, nitrogen, and sulfur complexes, HS facilitate global carbon and nitrogen cycling and the transport of anthropogenic contaminants. While it is known that HS also interact with organisms at different trophic levels to produce beneficial and harmful effects whether HS exert these biological effects through accumulation remains unknown. Current radiolabeling techniques, which only detect the amount of accumulated radiolabels, cannot visualize the transport and accumulation behavior of HS. Here, using a label-free method based on pump-probe microscopy, we show HS entered the protozoan Tetrahymena thermophila, zebrafish embryos, and human cells and exerted direct effects on these organisms. HS accumulated in the nucleus of T. thermophila, chorion pore canals of zebrafish embryos, and nucleus of intestinal and lung cells in a concentration- and time-dependent way. Epigenetic and transcriptomics assays show HS altered chromatin accessibility and gene transcription in T. thermophila. In zebrafish larvae, HS induced neurotoxicity, altering spontaneous muscle contraction and locomotor activity. Detailed images showing HS accumulation in our study reveal new insights on the ecological and environmental behavior of HS.

Unbound Natural Organic Matter Competes with Nanoparticles for Internalization Receptors During Cell Uptake.

Natural organic matter (NOM) that forms coronas on the surface of engineered nanoparticles (NPs) affects their stability, bio-uptake, and toxicity. After corona formation, a large amount of unbound NOM remains in the environment and their effects on organismal uptake of NPs remain unknown. Here, the effects of unbound NOM on the uptake of polyacrylate-coated hematite NPs (HemNPs) by the protozoan Tetrahymena thermophila were examined. HemNPs were well-dispersed without any detectable NOM adsorption. Kinetics experiments showed that unbound NOM decreased the uptake of HemNPs with greater inhibition at lower concentrations of the particles in the presence of NOM of higher molecular weight. The unbound NOM suppressed clathrin-mediated endocytosis but not the phagocytosis of HemNPs. Confirmation of these events was obtained using label-free hyperspectral stimulated Raman spectroscopy imaging and dissipative particle dynamics simulation. Overall, the present study demonstrates that unbound NOM can compete with HemNPs for internalization receptors on the surface of T. thermophila and inhibit particle uptake, highlighting the need to consider the direct effects of unbound NOM in bioapplication studies and in safety evaluations of NPs.

Algal Foods Reduce the Uptake of Hematite Nanoparticles by Downregulating Water Filtration in Daphnia magna.

Rapid developments in nanotechnology have led to the release of substantial amounts of nanoparticles (NPs) into aquatic environments, where many types of biotic particles are present and could potentially interact with the NPs. Nevertheless, how biotic particles may affect the bioaccumulation and toxicity of NPs remains largely unknown. In the present study, we investigated the effects of the green alga Chlamydomonas reinhardtii on the accumulation kinetics (uptake, assimilation, efflux) and toxicity of polyacrylate-coated hematite NPs (HemNPs), using Daphnia magna as the test organism. As a biotic particle and daphnid food, C. reinhardtii reduced the accumulation and toxicity of HemNPs in D. magna. The HemNPs were well-dispersed with little adsorption to the alga. Their decreased accumulation could thus be partly explained by their low trophic transfer from the alga to the daphnid and by the inductive effects of the alga on HemNP efflux. However, the main cause was the direct inhibition of HemNP uptake from the water phase as a result of the reduced water-filtration activity of D. magna in the presence of C. reinhardtii. Overall, in bioaccumulation studies, the inhibitory effects of biotic particles on NP uptake from the water phase should be paid attention.

A label-free technique to quantify and visualize gold nanoparticle accumulation at the single-cell level.

Despite their wide bioapplications, potential health risks of gold nanoparticles (AuNPs) remain unclear. As a determinant of their risks, AuNP accumulation within a cell population is subject to cell-to-cell heterogeneity. Methods to simultaneously quantify and visualize intracellular AuNPs at the single-cell level are, however, lacking. Here we developed a novel label-free technique, based on hyperspectral imaging with enhanced darkfield microscopy (HSI-DFM), to visualize and quantify AuNP accumulation at the single-cell level. The identification ability of the hyperspectral libraries derived from extra- and intracellular AuNPs was compared. The spectral number in the libraries was optimized to maximize their identification ability while minimizing the identification time. In addition, a filtration method was established to merge spectral libraries from different cell lines based on their similarity. The intracellularly accumulated AuNPs as determined by HSI-DFM well correlated with those detected by inductively coupled plasma mass spectrometry. This validation allowed us to calculate the intracellular concentration of AuNPs at the single-cell level and to monitor the accumulation kinetics of AuNPs in living cells. The label-free method developed herein can be applied to other types of AuNPs differing in their physicochemical properties as well as other NPs, as long as they are detectable by HSI-DFM.