LETTER pubs.acs.org/NanoLett Visualizing Gold Nanoparticle Uptake in Live Cells with Liquid Scanning Transmission Electron Microscopy Diana B. Peckys and Niels de Jonge* Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States ABSTRACT: The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a micro- ﬂuidic chamber and imaged using scanning transmission elec- tron microscopy. A quantitative image analysis showed that Au- NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ( 9 and 164 ( 4 NP-ﬁlled vesicles. KEYWORDS: Biological electron microscopy, live cells, scanning transmission electron microscopy, STEM, gold nanoparticles, nanotoxicology, nanoparticle uptake Certies with speciﬁc surface bound ligands, a combination that onjugated nanoparticles (NPs) join distinct nanoscale prop- has led to widespread applications in diagnostics and thera- peutics.1 4 NPs can for instance be used as eﬃcient contrast agents for molecular imaging,5 as carriers for targeted drug6 and gene delivery,7 and as therapeutical reagents for targeted photothermal therapy.8,9 Cytotoxicological studies of NPs are a major part of the assessment of NPs for medical applications and include the visualization and characterization of in vitro NP uptake.10 13 NP uptake is inﬂuenced by many factors, such as NP size, shape, surface chemistry, colloidal stability, nonspeciﬁc interactions, and cell line.14 20 Imaging studies provide crucial information on NP cell interactions, especially on the intracellular traﬃcking and fate of NPs and on their incorporated quantity.21 23 Single NPs cannot be resolved with light microscopy, for that reason, electron microscopy (EM) is used to record high-resolution images of intracellular NPs. Unfortunately, the sample preparation for EM typically includes processing of samples into 50 200 nm thin sections, a step that involves the risk of NP removal. A further disadvantage is the diﬃculty to quantitatively evaluate NP uptake from conventional EM images, since thin sections may be cut through three-dimen- sional structures, such as vesicles with high densities of NPs. Here, we present a new approach for the study of NP cell interactions. Live ﬁbroblast cells (COS-7) loaded with Au-NPs were imaged in liquid with scanning transmission electron microscopy (STEM) using a microﬂuidic chamber24,25 that provided a continuous ﬂow of buﬀer. STEM images were recorded through the transparent windows of the chamber; see Figure 1. The cells were alive at the onset of STEM imaging, and the obtained images thus provide information about the NP distribution in pristine cells. This EM approach can be used to study cells of up to ∼10 μm thickness, excluding the need for sectioning and requiring only a minimal sample preparation. The microﬂuidic chamber was made from two silicon micro- chips, each having a 50 nm thin silicon nitride (SiN) membrane in their center,25 providing transparent windows for photons and electrons at the energies used in this study. At the same time, the SiN membranes enclosed the liquid in the chamber and thus sealed the cells from the vacuum in the electron microscope. The dimensions of the SiN membranes were 50 200 μm or 50 400 μm. One microchip had a ﬂat surface and was used as substrate for the adhering cells; the second microchip had a 6 μm thick spacer in parallel with the two long sides. This spacer prevented compression of the cells when both microchips were assembled into a microﬂuidic chamber. The microchips were cleaned, and coated as previously described.24 Brieﬂy, the ﬂat microchips (without spacer) were rinsed in acetone followed by a rinse in ethanol (both from Sigma Aldrich) and then ambient air plasma cleaned. To promote attachment of the COS-7 cells, a coating with poly-L-lysine (PLL) (Sigma Aldrich) was applied. The COS-7 cells (green monkey kidney ﬁbroblast, from ATTC) were grown in Dulbecco’s Modiﬁed Eagle’s Medium (ATTC), with 10% fetal bovine serum (Invitrogen), at 37 C in a 5% CO2 environment. To grow cells directly on the microchips, tahdehecreelnlst wceelrlelahyaerrvewsittehd Cuap2oþn/M∼g920þ%frceoenDﬂuuelbneccycboy’s rpinhsoinspghtahtee buﬀered saline (GIBCO), incubating the cells with cell stripper solution (MediaTech) and subsequent quenching and suspen- sion in the medium. One droplet of cell suspension was added per PLL-coated microchip. Once a suﬃcient number of cells (3 12) had settled down on the SiN membrane, usually after a Received: Revised: Published: January 25, 2011 February 23, 2011 March 16, 2011 r 2011 American Chemical Society 1733 dx.doi.org/10.1021/nl200285r | Nano Lett. 2011, 11, 1733–1738