Response to “Stripy Nanoparticles Revisited”

In a paper entitled “Stripy Nanoparticles Revisited”, Lévy and co-workers contest the interpretation of scanning tunneling microscopy images of monolayer protected gold nanoparticles that our group has presented. We show that the two arguments they use are based on flawed assumptions and contradict each other. We also show new evidence for the existence of stripe-like domains on mixed monolayer-coated gold nanoparticles. Trasmission electron microscopy (TEM) and X-ray diffraction (XRD) data2 Fourier transform infrared (FTIR) spectroscopy studies3 Atomic force microscopy (AFM) images in water4 computational studies5, 6 In the latter case, a remarkable agreement both in terms of domain spacing5 and in terms of the existence of a size regime where these stripes occur6 was found. It was also established that these particles have a non-monotonic dependence of their saturation concentration (solubility)7 and their interfacial energy4 on their ligand shell composition, in good agreement with molecular dynamics simulations. They exhibit protein-resistance properties,2, 8 can penetrate cell membranes,8 and posses two polar point defects that can be selectively functionalized with a place-exchange reaction9 that has been modeled so as to calculate the free energy of the reaction.10 In ref. 1, Lévy and co-workers present a series of arguments questioning the interpretation of our data as well as some of our results. It appears that some parts of our work might be misinterpreted and hence we are grateful for this opportunity to clarify these aspects. Our experiments are no doubt challenging. Through the years, we have dedicated a good amount of time to improving our experimental approaches and to derive a more reliable picture of our system, both through experiments and through simulations. We appreciate every effort to stimulate a constructive scientific debate and hope that this exchange will contribute to a better understanding of self-assembled monolayers (SAMs) on nanoparticles as well as their investigation with scanning probe microscopy. The central argument presented in ref. 1 has to do with the interpretation of our STM images of mixed ligand nanoparticles, which Lévy and co-workers claim to be incorrect. We will present here a rebuttal to all of the points raised in ref. 1. In most cases we will start by correcting the selected presentation of our results given in ref. 1, to properly represent our body of work. We will also present new data to testify to our continuing research toward a deeper and more accurate interpretation of our STM data. In ref. 1, there exists the suggestion that our work on cell membrane penetration by striped nanoparticles cannot be reproduced; it should be stated up front that all of the data presented in our original publication8 have been reproduced in identical systems by our group,11 by our collaborators,12, 13 and in similar systems by groups not related to us.14 The main scientific argument developed in ref. 1 revolves around the interpretation of the STM images used to characterize our nanoparticles. We agree that this point merits an open discussion in the literature and we take this opportunity to deepen our discussion of the topic. Since our first publication,2 we have dedicated considerable attention to this point and are continuing to do so, as shown by the new results presented here. The authors of ref. 1 acknowledge our body of work on these particles, including four papers that deal almost entirely with STM images.2, 15-17 However, they choose to comment only on our first publication, ignoring answers available in the later ones. Science is a process, and it would be fair to assume that later papers offer a better understanding of a system and represent more accurately the position of a group of scientists. For the sake of clarity, we will first briefly restate the relevant results and conclusions that we have published over the last eight years and then proceed to answer the different points raised in ref. 1. In 2004, we presented a series of STM images whose interpretation was corroborated by XRD and TEM data.2 In these publications, we suggested that mixtures of dislike molecules spontaneously form striped domains on nanoparticles. In 2007, in a theory paper supported by experimental evidence, coarse grain and molecular dynamics simulations of our particles were presented. The simulations showed the presence of stripe-like domains with local order.5 Since then we have used in all of our publications a definition of our particles as containing “stripe-like” domains. We have since found a good number of structure–property relationships that link this particular configuration of ligands with macroscopic properties. None of these properties require a ‘perfect’ stripe alignment but only a local one. Representing our work as ‘perfect’ stripes on nanoparticles is a misrepresentation, and we hope that this communication will help to clarify this point. The simulation results shown in ref. 5 and the STM images presented in Figure 1 of ref. 15 (our second publication on this topic) show that our particles are made of stripe-like domains that are locally ordered. These are the representative images that we show in all of our presentations and proposals. Even in the case of our paper on the formation of chains of ‘striped’ nanoparticles via the functionalization of their polar defects,9 no ‘perfect’ alignment is required (as the authors of ref. 1 imply we state). For example, Figure 3d in ref. 5 shows that these poles also exist in the case of an imperfect ‘stripe-like’ arrangement. a) Molecular resolution STM topography image of hexanethiol-coated nanoparticles. b) Current image. c) Zoom of the delimited region of (a) showing headgroup arrangement. d,e) Height profiles along the dark lines in (a) together with Gaussian fits (dashed red lines) used to extract headgroup spacings (Bias voltage = 500 mV, Set current = 600 pA). The scan lines (1) and (2) (shown in (d) and (e), respectively) are taken on the middle and edge of a nanoparticle, respectively, yet they show identical spacing between the headgroups. The central claim of ref. 1 is based on two arguments: (1) that a simplified representation of striped particles would lead to a decreasing stripe width at the edges of our particles; (2) that Fourier transform (FT) analyses of our STM images evidence a periodicity in our images that is inconsistent with the presence of striped domains on nanoparticles. The former point is affected by a fundamental misunderstanding of the working principle of a scanning probe microscope, and can be disproved experimentally, while the latter is affected by a fundamental mistake in the interpretation of Fourier transforms of digitalized images, that can be readily identified through critical image analysis. The first argument in ref. 1 deals with what we believe is a nontrivial problem. Imaging curved surfaces in scanning probe microscopy is complex, as arguably the microscopes have been engineered to image mostly-flat samples. Figure 1 in ref. 1 does not capture the reality of STM image acquisition, as it suggests (without showing it) that the tip follows a horizontal, sample-independent trajectory onto which sample-derived features (tunneling currents) are projected. Hence, the central argument in Lévy's paper is based on two assumptions: (1) an STM tip moves horizontally on a sample, and (2) tunneling currents flow perfectly vertically from the tip into the substrate holding the sample. Both assumptions are invalid. In reality, the tip follows the contour of the sample.23 The correct projection of the sample features being imaged with STM is onto the true tip trajectory, not onto an imaginary flat line. If we assume a tip trajectory that maintains a constant distance from the particle's center of mass (hence making a semicircular trajectory), then images of the idealized particle shown in ref. 1 would be projected onto such a semicircle and consequently should show stripes with a spacing of ∼1 nm. It should be noted that, were the tip to really move horizontally over the sample, there would be no feedback needed, no feedback loop-artifact possible, and the whole interpretation of the images presented in ref. 1 (feedback loop artifacts) would be in contradiction with the initial argument. The second assumption in ref. 1, that is, directionality of the tunneling current, is also invalid as, while very little is known about the exact path the current will take from the STM tip to the substrate, there is much evidence suggesting that the current will tunnel into the gold cores of the nanoparticles first. Despite this, ref. 1 raises a valid point about imaging curved surfaces, given that the true tip and the true current trajectories are hard to determine. We have studied this problem in our group by analyzing images of homoligand nanoparticles. For these particles, our images show locally ordered patterns (of dots) that are simply an extension of the 2D crystals that the same molecules form on flat surfaces.18, 19 Hence, the interpretation of images of these particles is more straightforward than for mixed-ligand particles, providing an ideal reference system. We note that the arguments presented in ref. 1 should also apply to the intermolecular distance between adjacent ligand headgroups on homoligand nanoparticles; thus, according to the reasoning of ref. 10, such a distance should significantly decrease at the edges of particles (keeping the same proportionality, the spacing should decrease from 5 Å to 3 Å). As shown in Figure 6 of ref. 15 as well as in new data shown here in Figure 1, single molecules can be clearly identified both in the center and at the edges of nanoparticles, yet no distortions in the intermolecular spacing at the particle's edges is observed. These experimental observations disprove the main point in ref. 1. The representation of our particles given in ref. 1 also assumes that all of the ligands are very rigid, something in contradiction with our physical insight into the flexible nature of the ligand molecules that form the stripes.5 Importantly, it should be noted that Lévy and co-workers chose not to discuss the fact that, in most of the published images of particles with stripe-like domains, stripes are indeed clear only in the center of the nanoparticles with the edges showing unclear features; this can be observed for example in Figure 5a of ref. 17. These images—which, as discussed in ref. 17, are the majority of the images we have—are in fact not in contradiction with the argument presented in ref. 1. The second argument from ref. 1 is based on FT analysis carried out by the authors on two of our published STM images. Unfortunately, their arguments appear to be based on an erroneous interpretation of the FT results, suggesting that their conclusions are derived from an image-analysis artifact. The argument in ref. 1 is that an FT analysis of a single STM image from our work shows two lateral parallel bands. This, according to the authors of ref. 1, means that our images “have a defined wavelength, λx, along the fast scanning axis but no defined λy wavelength and therefore no overall wavelength λ”. As shown in Figure S1 in the Supporting Information, a series of aligned bands (with a very well-defined wavelength λ) shows two parallel bands in the FT, questioning this interpretation of the FT images. Furthermore, the authors of ref. 1 perform an FT analysis on an image that they artificially construct from a series of cartoons of idealized striped particles. They find that an FT analysis of their image yields circular features. They claim that the circularity of their features confirms their argument. They seem to suggest that the circularity in their images is due to a ±5° rotation in the alignment of their ‘idealized’ particles. As shown in Figure S2 of our Supporting Information, an FT of the same image with all of the ‘idealized’ particles aligned keeps showing the same circularity, disproving this point. These observations raise questions about the interpretation of the FT images. More questions can be raised by noticing that in the FT analysis of the STM image presented in ref. 1, a periodicity of 1.3 nm is found. This is in direct contradiction not only with our own measurements of average stripe width (based on 60 measurements) but also with those presented by Lévy and co-workers, in Table S1 of in ref. 1 (average width 1.08 ± 0.15 nm). Similar questions could be asked about the periodicity measured by the FT analysis of their cartoons, given the absence of an overall periodicity in those images. Hereafter, we show that these features have little to do with the periodicity in the images but mostly depend on the shape of the objects present in the images. It is well known that FTs of images containing large features (compared to the size of the image) will tend to produce patterns reflecting the shape of the feature (a circle for a circle, rectangles and ovals turned 90° for rectangles and ovals, respectively). Hence, a circular shape like the one used to represent striped particles in ref. 1 leads to a circular band in the FT image. This can be proven by performing transformations on the image that preserve the periodicity the authors of ref. 1 claim to be studying. As shown in Figure 2, the FT of the cartoon used by the authors to re-create ‘idealized’ striped nanoparticles yields a circular pattern (central images), as shown in ref. 1. If both ends of the nanoparticles in that image are clipped so as to make the object appear more rectangular, or if the image is stretched parallel to the direction of the stripes, the FT images result in rectangular and oval patterns, respectively. Given that in both cases the periodicity is not altered, the arguments used by the authors of ref. 1 have to be incorrect. Images of idealized particles (top row) with their FT analysis (middle row). The bottom row show the same images shown in the middle row with the shape highlighted in the top row images simply turned 90° as one would expect going from direct to reciprocal space. In the middle column is the cartoon and the FT image that Lévy and co-workers use to substantiate their claims, left and right are two variations on the image that do not affect the ‘periodicity’ in the image but lead to completely different FT patters. It is easy to interpret these images, as each image is by the FT of the shape of the particles that it by the more rectangular feature on the a circular on in the middle and an oval one on the images the same number of The features were to the images FT analysis. this one can two more of images. It is to take the ‘idealized’ images from ref. a ±5° in an to what is observed in the In such a case (as shown in Figure the FT shows indeed two parallel bands there is no a circular shape in the it is to take an STM image of one of our particles and it in a pattern similar to that shown in ref. 1. As in this case, the result is an oval shape in the FT that is the reciprocal image of the shape of the particles Figure the interpretation of the FT images in ref. 1 appears to be based on an image-analysis not related to the periodicity of our images. In ref. 1, the authors claim to a in images of our nanoparticles, this as a of our images. We should first that the images shown for example in and do not present this and also that this image feature can be simply as in the ‘stripe-like’ domains present on our particles, perfectly with our one cannot use these features to between different image 1 also that, in ref. the STM images of our particles do not the size of our particles in the same make this the authors use the average size of our particles their in ref. in ref. we not image with STM particles of the average size and the images are with the size in ref. we used a sample and showed that the STM size images do the of the particles shown The analysis presented in ref. 1 does not of the arguments used to substantiate our image We have based our on a series of and analyses whose we Through the years we have shown that there are between images of homoligand nanoparticles and those of particles with mixtures of as can be by Figure 1 to 3 and We images of locally aligned stripe-like domains The main has been to the tip and to show that the average stripe width is almost as a of tip We a analysis to and our 1 suggests that the images we are due to a feedback loop such as the of our tip on a In such a case, the width of the image stripes would depend on tip be at as shown in ref. 17. This is not the case for images of our particles, as proven by a analysis based on image and data This was by images of the same particles with and We have shown that the of particles showing stripes the same as images at tip were out STM images in on an of striped nanoparticles at different tip with average stripe width with average stripe width with average stripe width nm. Images in the middle row are particles to better show stripe-like domains, the bottom row show the same images with lines to the on the striped domains. STM images in on an of striped nanoparticles at two different scan and are of the shown in (a) and respectively. and are identical to and (e), the and the same particles with stripe-like domains, according to the image nanoparticles of the same show the same average stripe width over while particles of different show a different stripe width Figure 6 in ref. and Figure in ref. in Figure 3 and Figure we also show that striped images and to the we have presented through the years in with a can also be in on an a stripe width with similar particles presented four years Figure 3 and Figure also show that the stripes of nanoparticles are with tip and with scan We believe that these results cannot be in the of ref. Given the amount of work that we have to the interpretation of our images, it would seem fair to expect an interpretation to all of our and not a selected 1 does not the fact that simulations have been on mixed-ligand and have shown the formation of stripe-like domains. These simulations have all of the features that we on nanoparticles. For example, we have proven correct the that particles are indeed These simulations do not have a but a one. This is it is known that at the is and it in a configuration is at as from reality as a as discussed in ref. and 15 for particles, molecules tend to have a alignment that the nature of the particles. In ref. 1, on our about the of striped nanoparticles that the saturation concentration that we is if to their results. They use as reference a single on homoligand nanoparticles. The of to the particles they used and the the fact that it was not the in our does not to We can only that, through the years, we have that the more the particles are of molecules in their ligand shell the they 1 that there are between our on particle in and and particle in 2, and ref. particles, ref. particles, and ref. particles. would or should all of these particles the same in terms of and given that the molecules that are In we a that shows that striped nanoparticles with a of and are to penetrate cell in an In our we presence of the nanoparticles by showing a in images of as well as by free images in TEM 1 to acknowledge the existence of relevant TEM images and of their and that we are affected by in our microscopy This cannot be relevant since our nanoparticles and striped and that only in the molecular of the ligand from the homoligand used as a These nanoparticles have the same size and of the ligand and hence cannot be affected by as would affect every ref. 1 that our of cell lines cell and as well as our imaging are not for cell penetration by nanoparticles. In in to our Lévy and co-workers have most of the experimental we used in our initial work. we the claim in ref. 1 that are more for cell penetration experiments and published work showing the same results we with The authors of ref. 1 also claim that imaging is a better imaging than microscopy for our We also find that indeed is a work carried out in has the of striped nanoparticles with The results are highlighted in a published where we the presence of striped nanoparticles and show that can of of nanoparticles in as to microscopy that the time to a single most Importantly, the free of striped nanoparticles in the we show that the of these particles and can be used to calculate local we have shown that an penetration exists for nanoparticles, of cell lines and imaging These results the of our It should be that four have used our particles and reproduced our data. In the interpretation to our scanning tunneling images in ref. 1 does not for all of the arguments presented in our body of work and only on our first Importantly, the authors of ref. 1 large parts of our most of the relevant results. arguments are based on the understanding of image in scanning probe microscopy and a particular interpretation of Fourier transform analysis of our images that we to be We show evidence that their analysis is an of the image The paper to claim that some of our presented data are not yet the only experiments presented to this claim are carried out significantly different to those presented in our work carried out in the of was to our results. Supporting is available from the or from the We are grateful to for very and critical of the and to for the images of the homoligand nanoparticles. of to are published as are but not or They are made available as by the The is not for the or of by the than should be to the for the