Microscopy Microanalysis Microsc. Microanal. 20, 189–197, 2014 doi:10.1017/S1431927613013986 AND © MICROSCOPY SOCIETY OF AMERICA 2014 Gold Nanoparticle Uptake in Whole Cells in Liquid Examined by Environmental Scanning Electron Microscopy Diana B. Peckys 1, * and Niels de Jonge 1,2 1 2 Innovative Electron Microscopy Group, INM-Leibniz Institute for New Materials, 66123 Saarbrücken, Germany Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232, USA Abstract: The size of gold nanoparticles ~AuNPs! can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy ~EM! is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 ~human lung carcinoma! cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ;80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter. Key words: whole cells, environmental scanning electron microscopy, scanning transmission electron microscopy, gold nanoparticles, lysosomes, nanoparticle uptake, nanoparticle size I NTR ODUCTION Gold nanoparticles ~AuNPs! are currently extensively investigated for applications in diagnostics and therapeutics ~Huang et al., 2007; Ghosh et al., 2008; Murphy et al., 2008; Thakor et al., 2011; Weintraub, 2013!. Many factors are known to influence the interaction of cells with AuNPs, ranging from cell characteristics and properties of the examined AuNPs, to the particular experimental design. Yet, various aspects of the cellular responses are not well understood ~Alkilany & Murphy, 2010; Lévy et al., 2010!, especially for processes at later stages, i.e. occurring after 24 h, when AuNPs are mainly accumulated in lysosomes ~Shukla et al., 2005; Chithrani & Chan, 2007; Hosta et al., 2008; Krpetić et al., 2010; Ma et al., 2011!. An important question is how one of the most important physico-chemical properties of AuNPs, their size, affects the uptake, storage, and transport processes ~Chithrani et al., 2006; Chithrani & Chan, 2007; Pan et al., 2007; De Jong et al., 2008; Nativo et al., 2008!. Light microscopy is often used to study organelles that interact with the AuNPs, and clusters of AuNPs in cells ~Wang & Ma, 2009; Rosman et al., 2012!, but due to the diffraction-limited resolution nanoscale dimensions cannot be resolved. EM is the best option for identifying the intracellular locations of AuNPs while being able to vizualize individual particles, but most studies involve the sectioning of cells, and, as a consequence, data is typically obtained from one or few cells and the sectioning may introduce Received June 27, 2013; accepted November 6, 2013 *Corresponding author. E-mail: diana.peckys@inm-gmbh.de various artifacts ~Brandenberger et al., 2010; Rosman et al., 2012!. New microscopy methods are needed that combine three desirable attributes for the examination of AuNP-cell interactions, namely nanoscale-resolution, high sample thoughput such that data of at least several tens of cells can be acquired and analyzed, and the ability to acquire quantitative data. As a model for respiratory AuNP uptake we chose A549 cells ~human alveolar epithelial cancer cells!. The cells were examined at early ~20 min! and late ~1 or 2 days! stages after AuNP uptake. We studied the influence of the AuNP diameter on intracellular uptake and storage of serum-proteincoated AuNPs. A total of 145 cells were imaged and the distribution of a large number of AuNP clusters was quantitatively analyzed. M ATERIALS AND M ETHODS Preparation of AuNPs Serum proteins were noncovalently bound to the surfaces of AuNPs. Aliquots of purchased stock solutions of 10, 15, and 30 nm diameter unconjugated citrate-coated AuNPs with respective concentrations of 9.3 nM, 2.3 nM, and 0.3 nM ~BBI International, Cardiff, UK! were centrifuged at 8,000 ! g. The AuNP pellets were resuspended in Dulbecco’s Modified Eagle’s Medium ~DMEM! ~Gibco, Life Technologies, USA! supplemented with 10% fetal bovine serum ~FBS! ~PAN Biotech, Germany!, and incubated at 378C with shaking for 1 h. The centrifugation step was repeated and the AuNP pellets were again dissolved in DMEM/10% FBS 190 Diana B. Peckys and Niels de Jonge to reach final concentrations of approximately 9 nM, 7 nM, and 3 nM for the diameters 10, 15, and 30 nm, respectively. All AuNPs solutions were prepared immediately before starting the AuNP incubation of the cells. Cell Culture and AuNP Uptake Experiments A549 cells ~from the German Collection of Microorganisms and Cell Culture, Braunschweig, Germany! were grown in DMEM, supplemented with 10% FBS, and 4 mM l-glutamine ~Greiner Bio-One, Frickenhausen, Germany! at 378C and 9% CO2 . After reaching 70–90% confluence the cells were detached ~cell stripper, from Mediatech, Herndon, VA, USA!, resuspended in DMEM with 10% FBS, and seeded on flat and electron transparent support microchips ~Protochips Inc., Raleigh, NC, USA! with 50 nm thin silicon nitride ~SiN! membranes of different dimensions ~50 ! 100, 50 ! 200, or 70 ! 300 mm! in their center. After 5–10 min the microchips with the adhering cells were transferred into new DMEM with 10% FBS and incubated over night at 378C in a 9% CO2 environment. A detailed description of the microchips and the procedures used to prepare them for cell attachment can be found elsewhere ~Ring et al., 2011!. Before the incubation with AuNP solutions the cells were serum starved for 2 h in order to enhance the uptake of serum-protein-coated AuNPs ~Chithrani et al., 2006; Siegwart et al., 2009!. The early phase of cellular AuNP uptake was studied by placing the microchips with the serum-starved, adhering cells in the respective AuNP solution for 5 min. Microchips were individually incubated with 14 mL droplets of the AuNP solutions in an upside-down orientation ~Ring et al., 2011! to exclude any effect of sedimentation of AuNP on the cellular uptake ~Cho et al., 2011!. Subsequently, the chips were rinsed twice in DMEM and incubated for another 15 min in DMEM with 10% FBS, at 378C and 9% CO2 . Experiments for the study of longterm ~.24 h! storage of AuNPs used an AuNP uptake time of 2 h, followed by the same rinsing steps and an elongated reincubation phase, at 378C in the CO2 incubator, for 22 h or 43 h. After this chase period without AuNPs, the cells were rinsed twice in phosphate buffered solution ~PBS!, and once in 0.1 M cacodylate buffer ~CB! with 0.1 M sucrose, pH 7.4 CB, before fixing with 3% glutaraldehyde and 0.1% tannic acid in CB, pH 7.4, for 10 min at room temperature. After rinsing once with CB and twice with PBS, the cells were incubated in 100 mM glycine in PBS for 2 min followed by rinsing twice with PBS. The fixation of lipids with osmium tetroxide was then done by incubating the cells for 30 min in 0.01–0.02% OsO4 in CB at room temperature, which was followed by a triple rinse in PBS. The cells were finally stored in PBS at 48C until imaging, usually done within 24–48 h. ESEM Imaging of the whole cells in fully hydrated state was done with an ESEM ~Quanta 400 FEG, FEI, USA! equipped with a STEM detector ~Bogner et al., 2005!. The stage contained a special sample holder, including a Peltier cooling element, and a solid state detector, mounted underneath the sample serving as a STEM detector. A gaseous secondary electron detector ~GSED! mounted at the pole piece ~above the sample! was used to record a parallel set of images, serving mainly as a control for the thin water film on the cells. The stage temperature was kept at 38C, and a pressure of 740 Pa was chosen in order to create a saturated water vapor atmosphere ~" 100% relative humidity! in the ESEM chamber ensuring the constant coverage of the cells under a thin film of water. Prior to sample loading into the ESEM stage, a microchip with cells was taken out of the cooled PBS, rinsed twice in ice cold ultrapure water, briefly blotted on the backside, and placed into the precooled Peltier stage ~38C!. Continuous visual control of the upper side of the sample during the transfer confirmed that the cells remained wet ~shiny surface!. As soon as the sample was fixed in the stage ~1–2 min after the blotting! it was wetted again with 3 mL of ice cold, ultrapure water. To prevent drying of the cells during filling of the specimen chamber with saturated water vapor, a purging sequence that controlled evaporation from and condensation on the sample was initiated ~Bogner et al., 2005; Kirk et al., 2009!. The sequence consisted of a fivefold cycling of the chamber pressure between 800 and 1,300 Pa, finishing at 800 Pa. The beam valve was opened and the ESEM at low magnification was used to locate the SiN membrane window with the GSED detector. After positioning of the SiN window the water layer was thinned by a stepwise reduction of the pressure to a final value of 740 Pa, taking about 3 min. As soon as the water film was thin enough an image in the STEM detector became visible, but not in the GSED. After further evaporation of the water layer, the GSED finally showed a signal of the cell and the AuNPs. Imaging was done with 30 kV of electron beam energy, a spot size of 3, and a working distance set between 6.8 and 7.4 mm. These settings resulted in a theoretical spot size of ;1.0 nm and a probe current of 0.6 nA. Images were recorded with bright-field STEM mode using pixel-dwell times of 30 or 50 ms, an image size of 1,024 ! 884 pixels, and magnifications up to 60,000!. Images shown are selected regions from the original images. R ESULTS Incubating the Cells with AuNPs The influence of AuNP size on cellular uptake and storage was tested in A549 cells with citrate stabilized and subsequently serum-protein-coated AuNPs of 10, 15, and 30 nm diameter, after short or long pulse-chase times. The cells were incubated with a solution containing AuNPs for a certain amount of time, referred to as the pulse period. The AuNP-containing solution was then replaced by cell culture medium, and the cells were grown for another period of time, i.e., the chase period. The pulse-chase protocol was applied to ensure that all intracellular AuNPs were examined at a similar stage of their uptake, transport, or storage. For short-term experiments, the pulse period amounted to Examination of Gold Nanoparticle Uptake in Whole Cells in Liquid 5 min, followed by a 15 min chase period in usual cell culture medium without AuNPs. For the study of longer uptake phases, the pulse duration lasted 2 h, and the chase period was 22 or 43 h. The times used in a particular experiment are indicated as, for example, 2 # 22 h for 2 h pulse and 22 h chase in the following text. ESEM of Early Stages of AuNP Uptake Imaging of the fully hydrated, whole cells, covered with a thin layer of ultrapure water, was done with ESEM STEM ~Fig. 1!. The STEM detector was employed to obtain high contrast on the AuNPs within the whole cells ~de Jonge et al., 2009a!. The hydrated state of the cells was preserved during imaging by choosing suitable temperature and pressure settings for the specimen chamber such that a thin layer of water covered the cells ~Stokes et al., 2003; Bogner et al., 2005; Muscariello et al., 2005!. The presence of a water layer was verified by comparing the signal from the STEM detector with that of the GSED. Example images of cells from the early stages of AuNP uptake and transport ~after 5 # 15 min! are shown in Figure 2 for 30 nm diameter AuNPs. Figures 2A and 2B are bright-field STEM image of A549 cells recorded at 10,000! magnification. Figure 2A shows two neighboring cells with evident cell borders on a bright background; the cells are individually marked with dashed lines as visual guides. Figure 2B depicts a region taken from a lamellipodium ~i.e., a projection of the mobile edge! of another cell. In both images small and dark spots ~examples are marked with colored arrow heads in Fig. 2A! are visible on top of diffuse intracellular structures. The spots represent AuNPs scattered all over the cell. Since they appear as sharp objects, we conclude that their vertical locations must have been similar within the focal depth of the ESEM. The AuNPs were thus presumably located at the cell membrane or were already internalized but resided in close proximity to the membrane. Figures 2A and 2B reveal that AuNPs can be found in three different formations: as singles or pairs ~purple arrow heads!, in clusters ,200 nm in size ~light blue arrow heads!, and in clusters of a size between 200 nm and 1 mm ~dark blue arrow heads!. Figure 2C shows an image recorded of the asterisk-labeled area in Figure 2A. At a magnification of 30,000! this image displays how individual AuNPs arrange into two large clusters and two pairs of AuNPs at the right side of the image. In the following paragraphs all error values represent the standard deviation. Inspection of 31 images recorded from six cells with at least 10,000! magnification revealed 50 single or paired AuNPs, 107 clusters ,200 nm ~average size: 102 6 36 nm, consisting of 4 6 1 AuNPs!, and 90 clusters .200 nm ~average size: 397 6 165 nm, containing an average of 34 6 20 AuNPs!. Thus 58% of the AuNPs were found in large clusters, 32% in small clusters, and only 10% were monodispersed or appeared in pairs ~see Fig. 2D!. Note that in addition to the small but prominent AuNPs, the STEM images show a second class of structures that exhibited a lower electron density than the AuNPs ~exam- 191 ples are marked with arrows in Figs. 2A, 2B!. These structures are interpreted to be lamellar bodies ~LBs!, referred to as specific organelle markers for lung alveolar cells ~Foster et al., 1998!. The LBs appeared as round submicrometersized organelles with a homogeously contrasted ~due to the osmium fixation! body, the surfactant storage compartment, surrounded by a shell of less electron dense material. Longer-Term AuNP Uptake The longer-term fate of the uptaken AuNPs was studied with AuNPs of 10, 15, and 30 nm diameter, all coated with serum proteins. Figure 3A shows a group of cells after the 22 h chase with 30 nm diameter AuNPs. The cells had grown into a confluent layer ~individual cells are delineated with dashed lines!. All cells contain scattered, dark round spots ~examples are indicated with white arrow heads!, clearly different from LBs ~indicated with arrows in Figs. 3A and 3D for comparison!. The abundance and density of these spots varied between the individual cells. A second image ~Fig. 3B!, recorded at a magnification of 40,000! at the star-labeled area in Figure 3A reveals that individual AuNPs were homogeneously localized within the round structures. We refer to these structures as vesicles since uptaken AuNPs of diameters !10 nm have repeatedly been shown to remain trapped in vesicles ~Bright et al., 1997; Chithrani et al., 2006; Brandenberger et al., 2010!. Note that the limiting vesicle membrane itself cannot be seen in these whole cell samples, because the used osmium concentration was too low to contrast membranes, higher concentrations would have obscured the intravesicular AuNPs. The typical distribution features, i.e., the fairly homogenous AuNPs distribution within a given vesicle and the scattering of the vesicles throughout the cytosol, remained unchanged when cells were examined after a total pulsechase time of 45 h. These features reflect the typical AuNP distribution found in all long-term experiments, and they were thus independent of the size of the AuNPs or the presence of a serum protein coat. Images of vesicles with 15 nm diameter AuNPs after 2 # 22 h, and with 10 nm AuNPs after 2 # 43 h are shown in in Figures 3C and 3D, respectively. Measurement of AuNP-Filled Vesicles In order to quantify a possible influence of the two experimental parameters, AuNP diameter and chase time ~22 or 43 h!, on the intracellular AuNP storage process, a total of 139 cells was imaged, and the sizes were determined of 1,041 AuNP-filled vesicles in ESEM-STEM images recorded at magnifications of !7,000!. Figure 4A illustrates this measuring procedure for an image recorded at 14,000! magnification after a 22 h chase from cells with 15 nm diameter AuNPs. Figure 4B shows the average vesicle size for all three sizes of AuNPs measured after chase times of 22 and 43 h. A significant increase in vesicle size is apparent between both groups of the 30 nm AuNPs and those of 10 or 15 nm diameter AuNPs ~indicated in Fig. 4B with *** reflecting p , 0.001 of the Student’s t test!. It can also be 192 Diana B. Peckys and Niels de Jonge Figure 1. Schematic representation of the imaging of internalized gold nanoarticles ~AuNPs! in a whole cell in wet state using environmental scanning electron microscopy with a scanning transmission electron microscopy ~STEM! detector. Cells were grown on thin silicon nitride membranes, and remained under a thin film of ultrapure water at all times. Electrons from the scanning electron beam transmitted through the sample to reach the STEM detector underneath the sample, while electrons above the sample were detected by the gaseous secondary electron detector. Figure 2. ESEM-STEM images of cells containing AuNPs for the early stages of their cellular uptake ~5 min pulse and 15 min chase, see text!. A, B: Two examples of images taken from cellular regions with adhering or just uptaken serumprotein-coated AuNPs of 30 nm diameter, appearing as small black dots @the two adjacent cells in ~A! are delineated with dashed lines#. Examples of single or paired AuNPs are highlighted with purple triangles. Examples of clusters ,200 nm and .200 nm in size are highlighted with light and dark blue triangles, respectively. Examples of stained laminar bodies ~LB!, typical for alveolar type II cells, are indicated with arrows. The magnification ~M! was 10,000!. C: “Close up” image of the area labeled with an asterisk in ~A!. Individually discernable AuNPs are found in clusters, suggesting their uptake in early endosomes ~M " 30,000!!. D: Circular distribution diagram of the three AuNP distribution patterns. ESEM, environmental scanning electron microscopy; STEM, scanning transmission electron microscopy. Examination of Gold Nanoparticle Uptake in Whole Cells in Liquid 193 Figure 3. ESEM-STEM images of AuNPs at later stages of the cellular uptake ~2 h pulse and 22 h chase!. A: Confluent cells containing 30 nm diameter AuNPs; M " 2,500!. B: Image of the region marked with a star in ~A!; M " 40,000!. The AuNPs are homogenously distributed within clusters, presumably lysosomes. C: Image of 15 nm diameter AuNPs after 2 # 22 h. M " 50,000!. D: Image of 10 nm diameter AuNPs after 2 # 43 h; M " 30,000!. ESEM, environmental scanning electron microscopy; STEM, scanning transmission electron microscopy. concluded that the 43 h chase led to the same average vesicle sizes in one size group as the 22 h chase. Therefore, the data from both time groups were merged for further analysis of the dependence of the vesicle size on the AuNP diameter. A vesicle size distribution histogram is given in Figure 4C. All three distributions are roughly bell-shaped, but whereas distributions of vesicles with 10 and 15 nm AuNPs almost overlap, the curve for the 30 nm AuNPs is shifted toward larger diameter values. The data presented in this study were gained from 145 cells, facing a total analysis time of ;80 h, resulting from 25 h for experiments including sample preparation, 25 h of ESEM imaging time, 20 h for measurements with ImageJ, and 10 h for data analysis with Excel. Resolution and Radiation Damage A final experiment served to determine the achievable spatial resolution of ESEM-STEM of a whole cell, and to check for possible signs of radiation damage. Figure 5A shows a vesicle containing serum-protein-coated 30 nm diameter AuNPs after the 43 h chase. The image was recorded with a 60,000! magnification, such that the majority of AuNPs was individually recognizable. A line scan ~intensity versus position! was drawn over one of the AuNPs, and the 25– 75% rising edge of the signal peak ~shown in Fig. 5B! revealed a resolution ~Reimer & Kohl, 2008! of 7 nm. No signs of radiation damage became apparent during the ESEM analysis of 145 cells. The electron doses applied to record ESEM-STEM images with magnifications between 6,000 and 50,000! ranged between 4 and 247 e$/Å 2. These doses are in a much lower range than electron doses used to record images of plastic thin sections of cells, for example, using STEM tomography ~400–5,000 e$/Å 2 ! ~Sousa et al., 2011!. We did not observe differences in the characteristic shapes of the AuNP-containing vesicles when imaging at different electron doses. The graph in Figure 5B, for example, was recorded with an atypically high electron dose of 712 e$/Å 2. It depicts well-preserved characteristics of AuNP distributions, i.e., the homogenous distribution of the intravesicular AuNPs, and the nearly perfect round shape of the vesicle. The fixed and fully hydrated cells thus seem to have sufficient stability for ESEM-STEM imaging to allow the quantitative analysis of the AuNP clusters. We do not rule out the occurrence of sample shrinkage in the vertical direction upon extensive ESEM imaging but this type of radiation damage would not affect our results. D ISCUSSION The use of an ESEM allowed us to image intact cells in liquid with a maximum spatial resolution of 7 nm on the 194 Diana B. Peckys and Niels de Jonge Figure 4. Quantification of the vesicle sizes containing AuNPs for the six different experimental groups used for examining the intracellular storage at later stages. A: ESEM-STEM image depicting the procedure to measure the sizes of the vesicles. Vesicles that appeared too blurred, due to their remote location from the actual focal plane, were not measured; M " 14,000!. B: Bar graph of average vesicle sizes found for AuNP of diameters 10, 15, and 30 nm, and for different chase times. Blue " 10 nm ~24 h: n " 232; 45 h: n " 18!, red " 15 nm ~24 h: n " 161; 45 h: n " 63!, green " 30 nm ~24 h: n " 468; 45 h: n " 99!. C: Size distribution histogram of vesicles filled with AuNPs of different size. Green " 30 nm diameter, n " 569; blue " 10 nm diameter, n " 250; red " 15 nm diameter, n " 224. ESEM, environmental scanning electron microscopy; STEM, scanning transmission electron microscopy. AuNPs. The sample preparation was reduced to a minimum, consisting of chemical fixation, cooling, and rinsing in ultrapure water only, which is comparable to typical procedures for light microscopy. The amount of time needed for sample preparation and data acquisition was therefore comparable to that for light microscopy studies of cells, being a factor of 50–70 shorter than what is needed for usual EM analysis using thin sections ~Schrand et al., 2010!. As a result it was possible to acquire and process data from 145 cells. The images included all AuNPs in each vesicle, in contrast to transmisson electron microscopy studies using thin sections. Sectioning may lead to various complications regarding the quantitative analysis of NP uptake ~Tantra & Knight, 2010; Peckys & de Jonge, 2011!. A major difficulty results from the fact that the vesicular cross-sections do not occur exclusively in the equatorial plane but randomly. Therefore, the deduction of quantitative results involving, for example, the vesicle diameter, from sections requires mathematical processing leading to uncertainties in the estimated vesicle volumes ~Glavinovic et al., 1998! or vesicle AuNP occupancies ~Brandenberger et al., 2010!. An alternative to thin sections is the preparation of whole mount cells, involving dehydration and drying of cells ~Braet et al., 1997!. Such EM samples allow examination of the complete population of whole vesicles within a cell. Whole mount cells have been used for EM studies of vesicular trafficking ~Stoorvogel, 2008!, and proved suitable for the evaluation of intravesicular AuNP distributions ~Baudoin et al., 2013; Dukes et al., 2011!. However, the needed dehydration and drying processes typically result in 20–40% shrinkage, which can lead to additional artifacts such as membrane fractures ~Braet et al., 1997!, furthermore, the time consumption is about a factor of ten larger than for wet ESEM cell preparation. To date, three EM methods are available for imaging of whole cells in liquid. Wet SEM ~Thiberge et al., 2004! uses a sealable capsule to enclose and separate cells from the high vacuum of a conventional scanning electron microscope ~SEM!. Backscattered electrons can then be detected through the sealing membrane. The atmospheric scanning electron microscope ~ASEM! is an inverted SEM that allows observation of wet cells in an open dish from beneath while a confocal light microscope ~LM! can observe them from above ~Nishiyama et al., 2010!. Because both SEM methods detect backscattered electrons the maximum focal depth and the maximum resolution are restricted, however, the later can still outperform the maximum resolution of superresolution LM methods. A resolution down to 4 nm can be achieved by using liquid scanning transmission electron microscopy ~liquid STEM! ~de Jonge et al., 2009b!. Here, the cells are enclosed in a microfluididc device and imaged at a higher beam voltage of 200 kV using a liquid flow holder in an STEM. Blurring of signals from high atomic number materials and from locations in deeper cell regions is thus significantly reduced compared to wet SEM and ASEM. However, the special equipment of a liquid flow holder and a STEM are mandatory. Examination of Gold Nanoparticle Uptake in Whole Cells in Liquid 195 Figure 5. Measurement of the resolution achieved by ESEM-STEM of intracellular AuNPs. A: Image of AuNPs recorded using a pixel dwell time of 100 ms and M " 60,000!. A line scan was drawn over a 30 nm diameter AuNP. B: From the 25–75% rising edges of the signal peak of the line scan a resolution of 7 nm was measured. ESEM, environmental scanning electron microscopy; STEM, scanning transmission electron microscopy. The three above-mentionned EM methodologies use simple and fast sample preparations similar to those used for LM, the same advantage applies for our study. By imaging the cells with a STEM detector we achieve a maximum resolution that comes close to the resolution of liquid STEM, and, we are also able to detect AuNPs from deeper regions inside a cell. As the imaging is done in the environmental specimen chamber of the ESEM, which allows keeping the samples in saturated water vapor or in thin water layers, the need for a liquid flow holder is circumvented. ESEM-STEM images recorded at early stages ~i.e., after 5 min chase and 15 min pulse! of AuNP uptake, revealed that 90% of serum protein-coated AuNPs were arranged in clusters, whereby about two-third of these clustered AuNPs resided in clusters larger than 200 nm, and one-third in smaller clusters. The remaining 10% of the AuNPs were monodispersed or paired. The high degree of clustering at this time point is consistent with the general concept of endocytosis, including ligand-receptor binding, clathrinmediated endocytosis, and trafficking in early endosomes ~Goldstein et al., 1979; Gruenberg et al., 1989!. This type of endocytosis was demonstrated to play a dominant role for the uptake of AuNPs of similar sizes as used in this study ~Chithrani & Chan, 2007; Brandenberger et al., 2010; Iversen et al., 2011!. The later stages of intracellular trafficking were characterized in our study by intravesicular storage for all types of examined AuNPs. Considering the involved chase times, these vesicles were probably lysosomes ~Shukla et al., 2005; Chithrani & Chan, 2007; Hosta et al., 2008; Krpetić et al., 2010; Ma et al., 2011!. The vesicles were scattered throughout the cells with a distribution pattern clearly differing from the configuration found in an earlier study of AuNP uptake in COS7 cells, where after 1 day almost all of the AuNP-containing vesicles were found in a large cluster in proximity of the nucleus ~Peckys & de Jonge, 2011!. Different vesicle distribution patterns are cell type specific ~Bhattacharyya et al., 2010! and may be caused by differences in the stability of microtubules ~Barua & Rege, 2009!. The image of a vesicle containing AuNP represents a vertical projection through its entire three-dimensional volume. Remarkably, the AuNPs were rather homogeneously distributed in this projected image, and we suggest that the AuNPs were thus adhering to the vesicle membrane ~Peckys & de Jonge, 2011!. This homogeneous AuNP distribution is consistent with the pattern found in live COS7 cells imaged with STEM ~Peckys & de Jonge, 2011!, as well as in human skin carcinoma cells for transferrin receptor antibodyconjugated AuNPs ~Hopkins & Trowbridge, 1983!. The particulate character of the vesicular AuNP distribution implies that the biomolecular corona had been preserved for at least two days. In contrast, a filling of the whole vesicle volume with AuNPs would have resulted in a density gradient toward the center of the vesicle, which would have indicated the dissociation of the shielding protein coat from the AuNPs ~van Deurs et al., 1995; Bright et al., 1997!. Comparison of vesicle size distributions found for serum protein-coated AuNPs revealed that those with 30 nm diameter were in vesicles with a size that was on average 80 nm larger than the vesicles containing the 10 or 15 nm AuNPs ~see Fig. 5A!. So far the hypothesis of an influence of the AuNP diameter on the storing lysosome size was only supported by indirect estimates of the relative differences in sizes of populations of vesicles obtained via fluorescence microscopy ~Ma et al., 2011!. Our nanoscale resolution data reveals a distinct shift of 80 nm toward larger diameters in the size distribution histogram of 30 nm AuNP-storing lysosomes versus lysosomes filled with 10 or 15 nm AuNPs. Our data also provide information about the distribution profile of lysosome sizes. Lysosomes are the last intracellular organelle through which AuNPs will pass before eventually leaving the cell ~Chithrani & Chan, 2007!. Examining how different AuNPs characteristics can influence the situation in lysosomes is therefore of crucial relevance for the assessment of AuNPs designed to serve medical applications ~Alkilany & Murphy, 2010; Soenen et al., 2011; Dreaden et al., 2012!. Future studies can combine ESEM-STEM imaging with chemical 196 Diana B. Peckys and Niels de Jonge analysis, by imaging the sample first with Raman microscopy ~Cronholm et al., 2013! or with high resolution in electron microscopes equipped with spectrometers for energy-dispersive X-ray ~Leapman & Andrews, 1991! or electron energy-loss ~Aronova & Leapman, 2013!. In addition, fluorescent labeling and imaging of different cell organelles and structures can be performed before EM imaging ~Murphy et al., 2011! to concommitantly identify specific proteins and pathways that participate in NP uptake, trafficking, storage, and exocytosis. The identified regions of interest can then be studied with the ESEM-STEM method to detect colocalized AuNPs with high resolution. C ONCLUSIONS The usage of ESEM-STEM in combination with the preparation of whole cells in liquid allowed collection and quantitative analysis of nanoscale data from 145 whole cells within an exceptionally small amount of time of ;80 h. We applied this method to evaluate the influence of the diameter of uptaken AuNPs on their intracellular storage. A total of 1,041 AuNP clusters and vesicles-containing AuNPs from different experimental groups were measured and compared. Analysis of the nanoscaled data revealed that the AuNP storing lysosome size distribution was shifted by 80 nm toward larger values, for AuNPs with 30 nm diameter, compared to AuNPs of 10 and 15 nm diameter. The fast and high resolution imaging methodology of intracellular AuNPs in hydrated and whole cells presented here represents a new and helpful tool for the examination of nanoparticle-cell interactions and for the study of trafficking processes, especially in endosomal and lysosomal compartments. A CKNOWLEDGMENTS We thank M. Koch for help with the experiments, A. Kraegeloh for discussion and support of the experiments, Protochips Inc., NC, USA for providing the microchips with silicon nitride support windows, and E. Arzt for continuous support through INM. R EFER ENCES Alkilany, A.M. & Murphy, C.J. ~2010!. Toxicity and cellular uptake of gold nanoparticles: What we have learned so far? J Nanopart Res 12~7!, 2313–2333. Aronova, M.A. & Leapman, R.D. ~2013!. 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