Nano Letters microﬂuidic chamber kept their calcein ﬂuorescence intensity unchanged for the maximal tested time period of 2.5 h, indicating that the cells were alive for at least 2.5 h in the microﬂuidic chamber. Note that the cells lost their viability in the chamber within 15 min without the ﬂow of buﬀer. To examine if the enclosing process caused delayed cellular damage, we inspected several samples for cell division, i.e., mitosis, the occurrence of which is the most stringent criterion of cell viability. Because mitosis is temperature dependent and the liquid ﬂow holder had no internal heater, we reopened the microﬂuidic chamber and placed the microchip with the cells in a dish ﬁlled with CO2-independent medium (Invitrogen). The dish was transferred onto the stage of the microscope, equipped with an environmental system to ensure a constant temperature of 37 C and 100% humidiﬁed air. The total time the samples spent in the microﬂuidic chamber was reduced to 4 min, because the cells adhered to the spacer microchip after 5 min, causing cell rupture upon opening of the microﬂuidic chamber. During the following 53 h, a time-lapse series of phase-contrast images was recorded with 3 min intervals, revealing three mitosis events at the location of the SiN window. A mitosis that happened after 39 h is shown in the area of the dotted oval in Figure 2d f. Cells were also tested for signs of programmed cell death,26 i.e., apoptosis, by incubating the samples for 30 min in Hoechst 33342 (Invitrogen), 1 μg/mL in TB, a nuclear ﬂuorescent stain. None of the tested cells had developed signs of programmed cell death 53 h after opening of the microﬂuidic chamber. Similar results were obtained for two further samples. Cells kept in the microﬂuidic chamber were thus viable following the most stringent criterion of viability, namely, mitosis. After we had ensured that it was possible to keep the COS-7 cells alive and intact in the microﬂuidic chamber, we conducted STEM experiments to study the fate of NPs taken up by COS-7 cells. Microchips with adherent cells were serum-starved for 2 h to enhance the following uptake of serum protein coated Au- NPs.27 Thirty nanometer diameter unconjugated Au-NPs (Ted Pella) were coated with serum proteins by mixing 0.5 mL of the Au-NP stock solution with 2 mL of 10% FBS supplemented medium and subsequent incubation at 40 C under rotation (30 rpm), for 2 h. Such serum protein coating has been shown to prevent clustering of the Au-NPs and to favor their endocytotic uptake.17 The Au-NP solution was centrifuged for 6 min at 5000g and the pellet was dissolved in 10% FBS supplemented medium, to yield a ﬁnal volume of 60 μL with an Au-NP concentration of ∼1.8 nM. Twelve microliter droplets of this Au-NP solution were placed against the inner rim of cutoﬀ PCR tube lids. Microchips with serum-starved cells were individually placed in an inclined orientation against a droplet, such that the cells were submerged and upside down in the Au-NP solution and incubated for 2 h in a humid environment, at 37 C in the CO2 incubator. Subsequently, the samples were rinsed and incubated overnight in 10% FBS supplemented medium, at 37 C in the CO2 incubator. After 24 h, the cells were enclosed in the microﬂuidic chamber. Imaging with liquid STEM took place within 3 min after enclosure. The STEM (CM200, FEI Company, Oregon) was set to 200 kV, a beam semiangle R of 9 mrad, a pixel dwell time of 20 μs, a probe current of 0.16 nA, a magniﬁcation of 16000, a pixel size of 8.7 nm, an image size of 1024 1024 pixels (representing sample areas of 8.8 8.8 μm2), and an annular dark ﬁeld detector semiangle of 70 mrad. Panels a and b of Figure 3 show selected cell areas of 4.9 4.9 μm2 (Figure 3a) 7.4 7.4 μm2 (Figure 3b), depicting two representative intracellular clusters that consisted LETTER Figure 3. STEM of live cells in a microﬂuidic chamber, 24 h after incubation with Au-NPs. (a, b) Images of intracellular Au-NP aggregations in two diﬀerent cells, illustrating that Au-NPs had concentrated in three- dimensional clusters of vesicles densely ﬁlled with Au-NPs. (c) Detail of panel b showing the distribution of single NPs. Blurred Au-NPs are located outside the focus plane and illustrate the highly three-dimensional arrangement of the structures. (d) Normalized size distribution histogram of Au-NP ﬁlled vesicles shown in panel a (gray) and panel b (black). of vesicles with Au-NPs, appearing as dark spots. The average diameter of this type of cluster was measured from light micro- scopy images and was 4.6 ( 1 μm (n = 25), where the variation represents the standard measured sizes, and the measurement accuracy was 0.5 μm. The STEM images revealed that each cluster contained more than hundred round structures that were densely ﬁlled with Au-NPs. Due to their approximate round shapes and their ﬁlling with Au-NPs, we suggest that these structures are vesicles. Figure 3c is a detail of Figure 3b high- lighting the distribution of single NPs inside the vesicles. The STEM images display the Au-NP-ﬁlled vesicles with diﬀerent degrees of sharpness; this indicates that they were not on the same focal plane, but scattered over several micrometers in depth. The sharpest NP images can be found when these are in the focal plane and at a vertical location close to the top window, where the electron beam enters the sample. The electron probe is blurred toward layers deeper in the specimen due to a combination of geometric broadening and beam broad- ening on account of interactions of the electron beam with the specimen. A fraction of 63% of the probe current was scattered into the opening angle of the detector, from which we calculated the thickness of the liquid24 to be 10 ( 2 μm. The Au-NPs appear as black spots on account of this large liquid thickness, leading to a contrast reversal. For thinner liquids the Au-NPs appear as bright spots. The liquid thickness was thicker than what was expected on the basis of the spacer of 6 μm, and can be explained by a bulging of the SiN windows outward into the vacuum.25 STEM imaging of pristine cells is subject to radiation damage. Yet, the STEM images do not show signs of severe damage. The majority of the Au-NP-ﬁlled vesicles in Figure 3 had round or 1735 dx.doi.org/10.1021/nl200285r |Nano Lett. 2011, 11, 1733–1738