Nano Letters oval shapes, and the pattern of intravesicular Au-NPs appeared homogeneous. The cells were exposed to 2 104 electrons per scan pixel of a size of 8.7 nm, and the average electron dose was thus 3 e /Å2. The electron dose was a factor of 10 above the dose limit for EM above which subnanometer structural features become damaged in wet biological specimens,28 and a factor of 10 below the corresponding limit used in cryo-EM.29 Note that the local electron dose directly in the focal plane was higher, maximal 1.6 102 e /Å2 (for a diameter of the electron probe containing 50% of the current of 0.9 nm). But, only a small region of a cell was exposed to the higher dose, because electron beam scanning occurred with lines separated by the pixel size of 9 nm, and beam broadening rapidly decreases the radiation intensity in deeper layers.30 The absence of signs of severe radiation damage in the images and the fact that the average electron dose was smaller than the dose used in cryo-EM lead to the hypothesis that the ﬁrst image recorded on a cell can be used to assess the distribution of NPs inside a live cell. Figure 3 shows that the Au-NP ﬁlled endocytotic vesicles had accumulated into a large cluster. Similar conglomerations of NP- ﬁlled vesicles have been reported for several types of NPs in the vicinity of the endosomal recycling compartment (ERC), a peri- nuclear organelle involved in endosome recycling.19,31 33 To determine the number of Au-NP-ﬁlled vesicles per cluster, the original STEM images were analyzed, of which selected areas are shown in panels a and b of Figure 3. Vesicles were visually identiﬁed and counted if their contrast exceeded the background gray value by at least 30% and if their size was >100 nm. The counting was repeated four times per image resulting in a total of 117 ( 9 (cluster of Figure 3a) and 164 ( 4 (cluster of Figure 3b) NP-ﬁlled vesicles. We cannot exclude that several NP-ﬁlled vesicles might have been located outside the image area; therefore these numbers represent minimum values. This applies particularly for the cluster shown in Figure 3a, because beam blurring possibly prevented the identiﬁca- tion of additional vesicles. From vesicles that were in or close to the focal plane, we measured the average diameter to be 0.26 ( 0.05 μm (n = 63, Figure 3b; n = 30, Figure 3a). The diameters were measured from the STEM images using Image J (NIH). The distribution of vesicle sizes is depicted in Figure 3d and shows a maximum for vesicle diameters between 200 and 300 nm in both analyzed clusters. This size distribution histogram is similar to the size distribution of lysosomes.34,35 Since the endocytotic pathway shuttles cargo from vesicles to early endosomes to late endo- somes/multivesicular bodies and ﬁnally, for degradation, to lysosomes, we propose that 24 h after their intracellular uptake the Au-NPs had ended up in lysosomes. To assess the distribution of Au-NPs within the vesicles, we counted the Au-NPs in selected vesicular regions of 17 17 pixels (150 150 nm2). Figure 4a shows an example that represents such an area from the vesicle at arrow #1 in Figure 3b. Figure 4b shows a line-scan over a region with darker pixels, presumed to represent an Au-NP, having a full width at half-maximum of 28 ( 5 nm, which corresponds to the 30 nm diameter of the Au-NPs. Individual NPs can thus be recognized but the image is so pixelated that Au-NP counting is diﬃcult. To facilitate the counting of NPs, the image in Figure 3b was processed with Image J (NIH), ﬁrst by enlargement of a factor of 10 and then by ﬁltering with Gaussian blur with a radius of 1.5 pixels. In the ﬁltered image of Figure 4c the NPs can be recognized better than in the original image. In this example, a total of 12 Au-NPs was counted as indicated by the circles. An LETTER Figure 4. STEM image analysis to determine the density of Au-NPs in vesicles. (a) Selected region of 17 17 pixels at arrow #1 in Figure 3b. (b) A line scan was drawn over a darker region assumed to represent an Au-NP in (a) (see arrow). The full width at half-minimum was 28 ( 5 nm. (c) Filtered image used for the counting of Au-NPs (circles). (d) Selected region at arrow #2 in Figure 3b displaying 14 Au-NPs. example from another region is shown (image processed) in Figure 4d (arrow #2 in Figure 3b); here, we counted 14 Au-NPs. A total of 25 of such image sections were placed over vesicles and analyzed and yielded an average of 12 ( 2 Au-NPs. The degree of overlap of Au-NPs in these vesicular image areas was suﬃciently low, such that we could identify and count individual NPs, and we found a homogeneous distribution of the Au-NPs throughout the analyzed areas. This distribution points toward localization of the NPs at the membrane of the lysosomes, rather than to a complete ﬁlling of the entire intra- vesicular space. If this entire space would have been ﬁlled, we would expect to see a higher density of Au-NPs in the central parts of the vesicles compared to the periphery, which was not the case. The degree of coverage of the vesicle membrane by Au- NPs was determined as follows. The surface area of an average- sized vesicle was 0.21 ( 0.07 μm2 assuming a spherical shape. A 30 nm large gold sphere with a 5 nm thick layer of bound serum protein yields an eﬀective Au-NP diameter of 40 nm. On account of the curvature of the inner vesicle surface and assuming a monolayer of tightly packed Au-NPs, a single Au-NP would occupy an area of ∼50 50 nm2, and the theoretical maximum density is thus 400 Au-NPs/μm2 on the inner vesicle surface. Consequently, an average vesicle could hold a maximum of 85 ( 30 Au-NPs (Table 1). The actual number of Au-NPs in an image, projecting both the upper and lower halves of a vesicle, was 12 ( 2 in an area of 2 0.15 0.15 = 0.045 μm2. The actual density at the inner vesicle surface was thus 267 ( 44 Au-NPs/μm2, and a vesicle contained an average 57 ( 26 Au-NPs, which corresponds to 67 ( 11% occupancy of the available membrane area. The total number of Au-NPs in the lysosome cluster was estimated to be (9 ( 5) 103. The ﬁnding that Au-NPs are bound to the inner vesicular surface is supported by some studies of Au-NPs in intracellular 1736 dx.doi.org/10.1021/nl200285r |Nano Lett. 2011, 11, 1733–1738