Nano Letters Table 1. Quantitative Analysis of Au-NP Distributiona number of vesicles in cluster 164* ( 4 vesicle diameter (μm) 0.26* ( 0.05 estimated vesicle surface area (μm2) 0.21 ( 0.07 number of detected Au-NPs per 0.15 0.15 μm2 12* ( 2 of image density of Au-NPs/μm2 on vesicle membrane 267 ( 44 number of Au-NPs/vesicle 57 ( 26 theoretical max. number of Au-NPs/vesicle 85 ( 30 theoretical max. density of Au-NPs/μm2 on vesicle 400 Au-NPs/μm2 membrane occupancy of available vesicle membrane area 67 ( 11% total number of Au-NPs in vesicle cluster (9 ( 5) 103 a Values marked with an astereisk were directly determined from the STEM image of Figure 3b. vesicles using conventional EM images.20 However, there is a controversy about such a vesicular membrane bound location, and several other published EM images show intravascular NPs that form aggregates in the vesicles.13,36,37 In addition, EM images often depict only a few NPs per vesicle,18,37 39 possibly underestimating the occupancy of the available area by NPs. These discrepancies in the location and in the amount of vesicular NPs might relate to concerns about the sectioning of EM samples.21 First, one cannot be sure where a vesicle was cut through, which complicates quantitative studies. Second, EM sample preparation is prone to introduce artifacts40 hindering quantiﬁcation in NP uptake studies. The issue of artifacts is multifaceted. NPs may become displaced by the diamond knife,41 or may be washed away during sample preparation. Cell ﬁxation can redistribute NPs coated with cell-penetrating peptides into the nucleus,42 and translocate NPs with positively charged proteins across the cell membrane.43 Staining can introduce electron-dense nanosized aggregates that are diﬃcult to distin- guish from the NPs under investigation.36,44 Third, with con- ventional EM it is almost impossible to conduct a quantitative study on the scale of the entire cluster of vesicles. In order to do so, many images from a large number of sections would have to be analyzed.45 Liquid STEM, on the contrary, achieves single NP resolution on fully hydrated, nonﬁxed, intact cells that are living at the onset of imaging. The images contain signals from the full three-dimen- sional space of the cell, which can be used for quantitative studies of NP uptake and spatial distribution as demonstrated in this work. The whole cell analysis can be done on a much faster time scale (hours versus days) than possible with conventional EM,41 making the STEM approach suitable for routine measurements, needed for instance when systematic variations of NP materials and surface coating are included in the uptake study.21 The biological structure of the cells cannot be visualized with high resolution using electron microscopy of whole cells in liquid, because the contrast obtained on the carbon-based materials within water is too low. The cellular structure may be visualized using ﬂuorescent probes and light microscopy, which can be correlated with the STEM images. Future studies on cell NP interactions may well include such a ﬂuorophore labeling to identify speciﬁc proteins and pathways that participate in NP uptake, traﬃcking, storage, and exocytosis, by light microscopy prior to STEM imaging. We studied the intracellular uptake of 30 nm diameter Au-NPs in COS-7 cells with liquid STEM. The spatial distribution of NPs LETTER was determined in fully hydrated, pristine cells that were alive at the onset of imaging. On account of the absence of preparation of the cells into thin sections standard in EM, we were able to conduct a quantitative analysis of the NP distribution. The NPs were located in vesicles, possibly lysosomes. A total of 117 ( 9 and 164 ( 4 vesicles were found in two analyzed clusters after 24 h of incubation. The NPs were bound to the membranes of the vesicles, with 57 ( 26 Au-NPs/vesicle. About 67% of the available membrane surface area was covered with NPs. The total number of Au-NPs in the vesicle cluster was (9 ( 5) 103. We propose that liquid STEM of live cells can be used for quantitative studies of NP cell interactions. ’ AUTHOR INFORMATION Corresponding Author *E-mail: niels.de.jonge@vanderbilt.edu. ’ ACKNOWLEDGMENT We thank T. E. McKnight, P. Mazur, D. W. Piston, and Protochips, Inc., NC. STEM images were recorded at the SHaRE User Facility, sponsored by the Division of Scientiﬁc User Facilities, Oﬃce of Basic Energy Sciences, U.S. Department of Energy. Research was supported by Vanderbilt University Med- ical Center and by NIH Grants R01RR018470 (to P. Mazur) and 1R43EB008589 (to S. Mick). ’ REFERENCES (1) Petros, R. A.; DeSimone, J. M. Nat. Rev. Drug Discovery 2010, 9, 615–627. (2) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538–544. (3) McCarthy, J. 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