Three little (nano) controversies and their morals

This post is a translation of an article originally published in French in Médecine/Sciences. The Editorial of the same issue (also in French) by Pierre Corvol is entitled Scientific integrity: the need for a systemic approach (open access).  You can download a pdf of my article, or, read at the publisher’s website (subscription). For citation, please refer to the original article as follows:

Trois petites (nano) controverses et leurs morales; Raphaël Lévy; Med Sci (Paris), 33 8-9 (2017) 797-800; Publié en ligne : 18 septembre 2017; DOI:

« Selon que vous serez puissant ou misérable, les jugements de cour vous rendront blanc ou noir » [1] [Depending on your social height, The law will see your crime as black—or else as white.] Thus concludes the Fable, by Jean de La Fontaine, The Animals sick of the plague : the donkey, guilty of the theft of a few blades of grass, is condemned to death, whilst the Lion and other powerful animals guilty of much more serious crimes are treated to praise and flattery. It is tempting and comforting to think that scientific judgments are of an altogether different nature. Seen in this light, science would reside outside of power struggles and the few mishaps (mistakes, frauds, conflicts of interest) would be rapidly corrected since the reality of the material world would quickly come back to us through experimental results if we were to try to ignore it for too long. The truth is however very different. A large fraction of published scientific results cannot be reproduced. It is not a few mishaps but structural problems which affect the foundations of the scientific enterprise [2, 3]. Peer evaluations seems to encourage the publication of extraordinary stories in high impact factor journals rather than careful and rigorous experimental studies. Contradictory or “negative” data are rarely published: scientific journals are not really interested, and us, scientists, are not particularly motivated by publicly stating our doubts on the work of colleagues who could be in charge of evaluating our next article or grant application. It is particularly urgent to repair our knowledge production system because science is at the center of numerous challenges critical for the future of human beings and the planet. The (real) problems of reproducibility have already been harnessed by lobbies to attack the credibility of scientists [4]. After the election for president of the largest scientific and military power of the world of a man who denies climate change, is very positive about the use of the atomic bomb, and, more broadly wages an open war against science and truth [5], we have a paramount need for science to be open, robust, capable of defending its independence, integrity and universal values. This seems a distant prospect.

The near absence of critical discussion in the scientific literature in many areas of science could make us forget that controversies are an essential aspect of the quest for knowledge, allowing to identify weak points of experiments and theories, thus enabling to consolidate or invalidate them [6]. They are consubstantial to the scientific practice [7]. The analysis of controversies is also a tool to “symmetrically map” the actors to better understand the roles of individuals and social processes [8]. In this piece, I describe three recent controversies in my area of research: gold nanoparticles applied to biology and medicine. This is no “symmetric map”: I am not a neutral observer but a scientist active, to various degrees, in each of those. I am trying nevertheless to draw some lessons and suggestions to improve the ways we work as scientists.

Stripy Nanoparticles

In 2004, Francesco Stellacci’s group at the Massachusetts Institute of Technology (MIT) published in the prestigious journal Nature Materials an article describing gold nanoparticles covered by a mixture of two molecules that self-assemble to form stripes that are observed by scanning probe microscopy [9]. This article and the numerous other ones that will follow in the same journal and in others just as prestigious such as the Journal of the American Chemical Society [10], Science [11] and Proceedings of the National Academy of Sciences (PNAS) [12], suggest that, thanks to their stripes, these nanoparticles have unique properties in terms of wetting, self-organization, interaction with proteins, penetration in cells, with lots of potential applications for biomolecular sensing, or even drug delivery. These articles certainly contributes to the progress of their authors’ careers, but the stripes are an experimental artefact well known by users of scanning probe microscopy. How to explain then that more than 20 “stripy” articles were published between 2004 and 2012? It is obvious that specialists (and even enlightened amateurs) had identified the problem as early as 2004. Yet, the articles and reviews of that period show no sign of it. One now knows that Predrag Djuranovic has been the first to engage into a scientific investigation aiming at testing, and eventually, contesting, the evidence for the the existence of the stripes. In 2005, this rigorous and brave scientist was a student in Francesco Stellacci’s lab. His experimental results and numerical simulation showing how the stripes originate from a poorly adjusted feedback control system were unambiguous but MIT ensured that these results would remain secret [13]. In 2007, I submitted a technical comment responding to the Science article. This first attempt, limited in its scope to the Science article itself, was unsuccessful: Science did ask Francesco Stellacci to respond but then decided not to publish the exchange of views [14]. In 2008, a new article from the MIT group, again in Nature Materials, report that, thanks to their stripes, these nanoparticles can cross the cell membrane and directly access the cytosol [15]. This is accompanied by a commentary entitled “Particles slip cell security” [16]. After discussions with several of my students, we decide to propose a more exhaustive answer. A few months later, the article “Stripy Nanoparticles Revisited” is ready. It includes a new analysis of the stripy images concluding that the stripes are a scanning artefact as well as a critical discussion of the physicochemical and biological properties which, together with experimental results, contradict the claim of direct access to the interior of cells. The article is first submitted to Nature Materials (rejected), then NanoLetters (rejected), and, finally, Small… where it is published after an editorial process that lasted three years [17-19]. The publication of our article, in November 2012, does not end the controversy. Instead, it expands in the scientific literature (a little) and it also takes new forms (in particular on my blog and others [20-23]). Problems with the reuse of images in different publications emerge and eventually lead to two corrections ([12] and [15]). After a number of requests, Philip Moriarty and Julian Stirling (School of Physics and Astronomy, university of Nottingham, UK) are given access to the original data of the 2004 article. They demonstrate, among other things, that the stripes are present in the entire image, i.e. even between the gold nanoparticles [24], a conclusion still rejected by Francesco Stellacci [25].

Homeopathic nanoparticles

The laboratory of Molly Stevens at Imperial College is one of the most prestigious in the field of biomaterials. In 2012, two articles from the group relate the particularly interesting properties of nanoparticles for diagnostic applications. The first one, published in Nature Materials, reports a phenomenon which is entirely extraordinary in which the signal detected increases when the concentration of molecules to detect decreases (“inverse sensitivity”) [26]. Even more incredible, this phenomenon extends to the point where there is less than a molecule of enzyme, on average, in the volume under study. The second article published in Nature Nanotechnology goes further : no need for instruments, the detection of concentration of analytes in the same range is achieved thanks to a colour change visible with the naked eye [27]. Detailed critiques of these articles are available on the website PubPeer [28, 29] as well as in a preprint authored by Boris Barbour [29b]; the objections are both simple and profound but the authors have chosen not to respond. One can note that the Avogadro number includes lots of zeros (630 000 000 000 000 000 000 000) and that the detection of a macroscopic change of property due to the presence of a single molecule is therefore an achievement that requires extremely solid proofs. One of the posts on PubPeer indicates that someone contacted the Editor of Nature Nanotechnology in January 2013, but, four years later, no doubts are expressed on the journal website, in the traditional scientific literature nor in the newspapers that had covered this story (e.g. Le Monde and the Daily Mail) when it was initially published [30, 31].

Spherical Nucleic Acids

The laboratory of Chad Mirkin at Northwestern University (USA) is one of the most prestigious in the field of nanosciences applied to biology and medicine. One major theme of their research are the Spherical Nucleic Acids (SNAs), a term introduced by Mirkin to describe gold nanoparticles functionalised with DNA (or RNA) strands. These SNAs are supposed to have properties very different from linear DNA [32]. In particular, they can access the cytosol of live cells, where they could detect and regulate, the presence and quantity of mRNAs. One could ask why this solution did not appear during evolution : to access the cell machinery, viruses and bacteria would have only needed to package themselves within their genetic materials. The first articles (in Science [33], the Journal of the Americal Chemical Society [34], NanoLetters [35], ACS Nano [36]) proposing this surprising theory do no mention the mechanism of the SNAs into cells whatsover. The following one, e.g. [37], propose that the particles enter by endocytosis, but do not explain the mechanism by which the SNAs would escape endosomes. After several dozens of articles on this topic, the proportion of particles reaching the cytosol is still to be measured and reported (in spite of the fact that gold nanoparticles have been used since the 1950s to study intracellular trafficking; such a study would not be difficult). One article from the Mirkin group suggests that SNAs are degraded in the endosomes and that a “small unquantifiable portion escapes […]” [38]. Nevertheless, the particles are now commercially available under the name SmartFlares (Merck Millipore) to detect RNA inside cells. We have studied the entry of nanoparticles in cells and their ability to detect RNA. Given our difficult experience with the publication of Stripy Nanoparticle Revisited, we decided to adopt a different strategy. The project has been open and we have shared our results in quasi real time on our blog. In contradiction with the descriptions made by Mirkin and by Merck Millipore, we have observed that the SmartFlares were degraded in endosomes and were not able to detect mRNA.  Mirroring the tale of Predrag Djuranovic and the stripy nanoparticles, we were not the first to have doubts about the technology: Luke Armstrong, who had been in charge of developing the SmartFlares at Merck Millipore in California (before leaving the company) had reached the same conclusion [39]. To ensure speedy publication and transparency, we published our article on the (not so prestigious) ScienceOpen platform where peer review occurs after publication [40]. We invited comments by Mirkin to no avail. Another article by the same group in PNAS describe a new version of the SmartFlares [41]. Our analysis of the raw data (obtained after multiple insistent requests) show that the signal comes from endosomes. Our letter submitted to PNAS has been rejected by the editorial board because it “[did] not contribute significantly to the discussion of this paper” [42].


Access to raw data is essential and guaranteed by clear rules adopted by Universities, scientific journals and funding agencies. It is therefore generally possible to access data with some efforts. It is obviously preferable to publish data at the same time as the articles. This is already the norm for some categories of results and it should become generalised. Researchers should also adopt the Manifesto for reproducible research [43]. The tools are in place to improve the practice of science.

Evaluations of science and scientists must imperatively be based on a critical analysis of their work and the robustness or their results, not on the prestige of the institutions or journals. This requires a change of mind and a clear commitment from researchers who are in positions of power, i.e. everyone who features on promotion or recruitment committees. To say that an article is good because it has been published in a prestigious journal is a moral and logical error which needs to be challenged.

Institutions and scientific journals are not motivated by the quest for scientific truth. The decisisons taken by MIT (keeping Predrag Djuranovic’s findings secret), by Nature Materials (not publishing the exchange with Francesco Stellacci [14]), and by PNAS (not publishing [42]) have directly impacted progress of knowledge. These institutions have commendable principles but, in practice, they aim first at defending their reputation and finances [44]. The latter objective only partially aligns with scientific progress which requires rapid and open discussion of results and conclusions. The Worldwide Web, invented for the sharing of science, enables this discussion. Researchers therefore should embrace the following tools: 1) Pubpeer to comment on articles; 2) Preprints to publish rapidly, minimise the influence of editors, and, dissociate publication, i.e. sharing of information, from evaluation, i.e. peer review; 3) Social networks, e.g. Twitter and blogs, which constitute an ongoing scientific conference to discuss experiments, results, methods, analyses, and new publications.

Acknowledgements: I thank Marianne Noel (IFRIS) for her critical reading of this piece, and, Marianne Lévy for comments on the grammar and style [French version] very necessary after 14 years in an English-speaking country…

Conflicts of interest: The author declares that he has no conflict of interest related to this article.


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Guest post: SmartFlares fail to reflect their target transcripts levels

Czarnek&BeretaThis is a guest post by Maria Czarnek and Joanna Bereta, who have just published the following article in Scientific Reports entitled SmartFlares fail to reflect their target transcripts levels

We got the idea of using SmartFlare probes when working on generating knockout cells. In the era of CRISPR-Cas9 genome editing, the possibility of sorting out knockout cells based on their low target transcript content (mRNAs that contain premature stop codons are removed in a process called nonsense-mediated decay) instead of time-consuming testing of dozens or thousands of clones would be a great step forward. SmartFlare probes seemed to be just the ticket: no transfection, lysis or fixation needed; moreover, the probes were supposed to eventually leave the cells. We were full of hope as the first probes arrived. (more…)

Hot topics in Biochemistry

Every year our 3rd year Biochemistry students have to produce posters on hot topics in Biochemistry. And every year I ask colleagues to contribute some ideas for this list of topics. You can find last year’s list here. And you can contribute to this year’s list via Twitter or in the comments below.


At the 254th ACS conference in Washington DC

This is a guest post by Sumaira Ashraf.

Last month, I got the opportunity to attend and present my work at the 254th ACS conference held in Washington DC. The session was organized by Raphaël Lévy, Niveen Khashab, and Zhihong Nie. I presented preliminary data regarding my work on multimodal imaging probes for stem cell tracking. I enjoyed the conference but was disappointed by some of the talks. Many speakers  exaggerate their results to impress their audience and hype the worth of their work. I did wonder if it is productive to point out mistakes as no one seems interested in correcting them, but, instead, they might become your lifelong rivals. Here is an example. I know from practical experience that liposomes loaded with small molecule dyes cannot be used as long-term imaging modality because within 24 hrs of loading ~99% signal intensity is lost due to leakage from the liposomal cavity. But speakers presented quantitative imaging data based on these approaches. I wanted to point this problem out but I did not want to get involved in never ending discussion where no one will be ready to admit their errors. Eventually, I did not say anything.

Of course, my own work with polylelectrolyte capsules has some limitations as well: while they are good candidates for protecting imaging probes from intracellular degradation and intracellular species from potentially toxic probes, the limit of detection depends on the amount of contrast agent delivered which still remain low. In my current experiments, I need to overcome this limitation by modifying or choosing an alternate approach.

The Kavli lectures were really very informative… but I could not attend the full talk of the 2nd speaker because the air conditioning was set at a temperature so low that I could not tolerate it: I was forced to leave this interesting lecture. The industry exhibition downstairs the convention centre was massive and fun. I particularly liked the Bruker stall which had good explanation of NMR, mass spectrometers, etc., (covering theoretical and experimental data evaluation). There were other advantages to the visit of the exhibition: chocolates, bags, T- shirts, ice cream (prepared in liquid nitrogen within 3 minutes), lunch, free L’Oréal products, and lucky draw (especially when you win; I won a Swag Bag of merchandise from the ACS Store worth USD$75.00). I was lucky to see the solar eclipse through the cover glasses provided by the conference.

I missed the opportunity to visit the reflecting pool near Lincoln memorial and some interesting museums due to time limitation. I am looking forward to some forthcoming opportunity to see them.

I came back with some thoughts to work with new nanocomposites (recent work presented in one of the talk by Amit Joshi; based on doping materials rather than combining them inside big capsules or attaching via linkers) for multimodal imaging which might have potential to overcome the flaws associated with above mentioned materials. But I need to try… without trying one cannot be sure of anything.


Some readers might wonder why I am going on about this, so let me tell you: this is a considerably more important story than Stripy Nanoparticles Revisited. If, as I am arguing, some of this science is shaky, then it is not only the way we evaluate scientists and spend public money which are put into question, but the foundation of ongoing clinical trials.

Back to basics: in the section of Mirkin’s group PhD dissertation (previous post) that respond to our critique of their work on Spherical Nucleic Acid / SmartFlare / StickyFlare, they wrote the following:

Additionally, since the commercialization and sale of the nanoflare platform under the trade name Smartflare (Millipore), dozens of researchers around the world have participated in successful sequence-specific gene detection.[80]

Reference [80] correspond to six (half a dozen) articles, 80a to 80f (see below for details and links). Out of these six, only two are actual research papers, and, for both, the SmartFlares are a very minor addition to the work. Out of these two, only one is completely independent of Mirkin/EMD Millipore (the other one comes from Northwestern).

80a) is not primary research; it is an advertorial produced by EMD Millipore.

80b) is not primary research: it is a 300 words congress abstract (no figure). A follow up paper by the same group is discussed here.

80c) is a review and it is a collaboration between Northwestern (Mirkin’s University) and EMD Millipore. CoI statement from the paper: “D. Weldon is the R&D Manager at EMD Millipore responsible for the production of SmartFlares. Patents related to therapeutically targeting Nodal in tumor cells have been awarded to E.A. Seftor, R.E.B. Seftor, and M.J.C. Hendrix.

80d) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. The SmartFlare is a minor part of the article.

80e) is not primary research: it is an advertorial in a magazine funded by company advertising (including EMD Millipore in that very issue). The author is a journalist working for the magazine, not a practicing scientist.

80f) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. SmartFlares are a very minor part of the article. The authors are from Northwestern, i.e. Mirkin’s University.


A response from Chad Mirkin’s group

Well nearly. Possibly as close as we might get.

If you have followed the Spherical Nucleic Acid / SmartFlare / StickyFlare story on this blog, you will know that we have raised doubts about the endosomal escape of these nanoparticles which are supposed to reach the cytosol of cells where they could detect mRNAs. We have even published an article on this topic. The Mirkin group has developed the technology and it has been commercialized four years ago for the live cell detection of mRNAs by EMD Millipore.

One PNAS paper on the topic was Briley et al (see here for letter to PNAS and what happened to that letter). In his PhD dissertation (Briley, W. E. (2016), William Edward Briley respond to our criticism. I reproduce the relevant section below. One might note that there is an incredibly simple way to determine the localisation of these particles inside the cells: electron microscopy, a technique which has been used for this exact purpose for over 50 years. We have used it. The results were unambiguous.

2.3 Commentary on the Endosomal Escape of SNAs
Though the endosomal escape of SNA nanostructures such as the Nanoflare and stickyflare is evident based upon their ability to provide sequence-specific information regarding RNA levels and locations within cells, one researcher has concluded that SNAs cannot escape from endosomes.[75] That researcher [That’s me!] is ignoring the many papers now that use such architectures for sequence-specific cell-sorting experiments. Indeed, if these architectures, which are taken up by scavenger-receptor mediated endosomal pathways,[68a] do not escape the endosome, then it is difficult to understand the reports by the many groups who have documented the sequencespecific function of SNAs (all of which require endosomal escape), in antisense gene regulation,[12, 44-45, 53] siRNA gene regulation,[20, 68b, 76] nanoflare gene detection,[47, 67a, 67c, 77] and sticky-flare gene detection.[78] Perhaps the best demonstration of this sequence specificity is in the function of the multiplexed Nanoflare.[67a] This nanoconjugate, discussed in chapter 1, contains two targeting sequences specific to two different genes (actin and survivin). When cells treated with multiplexed nanoflares were subjected to siRNA that specifically knocked down the expression of survivin, a corresponding decrease of fluorescence was observed in the nanoflare’s survivin-associated fluorescence (cy5), but not the actin-associated fluorescence (cy3) compared to control.[67a] Likewise, when cells were subjected to actin-targeting siRNA, a decrease in actin-associated fluorescence was observed, with no decrease of survivin-associated fluorescence.
These results indicate that the detection by nanoflares is unique to the targeted mRNA transcript. To rule out any possible effects of the fluorophores, the Cy5 and Cy3 dyes were switched to the other gene (actin-cy5, survivin-cy3), and again the corresponding sequence-specific responses were observed. Importantly, since the development of the multiplexed nanoflare, other research groups have independently developed nanoflare-like structures capable of sequence-specific detection of 3,[57] and even 4[79] genes simultaneously. Additionally, since the commercialization and sale of the nanoflare platform under the trade name Smartflare (Millipore), dozens of researchers around the world have participated in successful sequence-specific gene detection.[80]
Further evidence of SNA endosome escape can be seen visually through analysis of the sticky-flare construct. If sticky-flares were incapable of escaping endosomes, one would expect consistent colocalization with endosomes. However, this is not the case. The patterns of β-actin targeted sticky-flares, when used in HeLa cells, instead exhibit localization around mitochondria. If such structures were limited to endosomes, it is inconceivable that they would co-localize with specific organelles. This is consistent with mitochondrial localization of multiple RNAs which
has also been identified by other groups, using multiple techniques, in HeLa cells.[74] Sticky-flare release from the endosome was further confirmed by designing sticky-flares targeting a second sequence, a U1 short nuclear RNA (snRNA). U1 snRNA is known to traffic from the cytoplasm to the nucleus. Indeed, cells treated with U1-targeting sticky-flares exhibited specific fluorescence within the nucleus. The pattern observed indicates nuclear localization through endosomal escape and sequence-specific tagging of an RNA that is actively transported into the nucleus. Again, this would not be possible if such structures were confined exclusively to endosomes. Speculation that SNAs do not escape endosomes has been fueled by the observation that the fluorescence pattern of β-actin in MEFs is punctate, which has been interpreted as an indication that the sticky-flares are simply trapped in endosomes. However, β-actin is well known to exhibit punctate fluorescence in many cases, an observation made by others in multiple cell lines, including MEFs.[81] Punctate fluorescence is very common in RNA-labeling studies and well known by researchers familiar with FISH.82 This is due to the fact that RNA is often packaged into large RNA-containing granules, which facilitates transport and translational control of the included transcripts.[82b, 83] Such packaging into granules has been extensively studied using β-actin mRNA. Thus, the fact that sticky-flares targeting β-actin were packaged into granules as previously observed, while U1-targeting sticky-flares were specific to the nucleus in the same cell line, demonstrates the functionality of the construct.
Taken together, the success of the many groups who use flare architectures for the detection and knockdown of RNA in cells, and the work of dozens of labs studying related nanoparticle constructs, provide unambiguous evidence of the ability for such architectures to escape the endosome and participate in reactions exclusive to the cytosol. The mechanism of endosomal escape for nanoparticle-based vehicles is currently unknown and is an interesting and important question that is actively being investigated by many in the field.[84]


Briley, W. E. (2016). Investigation and manipulation of the local microenvironment of spherical nucleic acid nanoconjugates (Order No. 10117274). Available from ProQuest Dissertations & Theses Global. (1795522748). Retrieved from

Quantum dots for Immunofluorescence

Guest post by Dave Mason

In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in combination with light microscopy, this makes for a versatile platform for research and diagnostics.

Most small-dye based fluorophores that are used in combination with antibodies suffer from a limitation; hit them with enough light and you irreversibly damage the fluorochrome, rendering the dye ‘invisible’ or photobleached. This property is the basis of several biophysical techniques such as Fluorescence Recovery After Photobleaching (FRAP) but for routine imaging it is largely an unwanted property.

Over 20 years ago, a new class of fluorescent conjugate was introduced in the form of Quantum Dots (QDots); semiconductor nanocrystals that promised increased brightness, a broad excitation and narrow emission band (good when using multi-channel imaging) and most importantly: no photobleaching. They were hailed as a game changer: “When the methods are worked out, they’ll be used instantly” (ref). With the expectation that they would “…soon be a standard biological tool” (ref).

So what happened? Check the published literature or walk into any imaging lab today and you’ll find antibodies conjugated to all manner of small dyes from FITC and rhodamine to Cyanine and Alexa dyes. Rarely will you find QDot-conjugated antibodies used despite them being commercially available. Why would people shun a technology that seemingly provides so many advantages?

Based on some strange observations, when trying to use QDot-conjugated antibodies, Jen Francis, investigated this phenomenon more closely, systematically labelling different cellular targets with Quantum dots and traditional small molecule dyes.


Figure 3 from doi:10.3762/bjnano.8.125 shows Tubulin simultaneously labelled with small fluorescent dye (A) and QDots (B). Overlay shows Qdot in green and A488 in Magenta. See paper for more details. See UPDATE below.

The work published in the Beilstein Journal of Nanotechnology (doi: 10.3762/bjnano.8.125) demonstrates a surprising finding. Some targets in the cell such as tubulin (the ‘gold standard’ for QDot labelling) label just as well with the QDot as with the dye (see above). Others however, including nuclear and some focal adhesion targets would only label with the organic dye.


The important question of course is: why the difference in labelling when using Quantum Dots or dyes? This is discussed in more detail in the paper but one explanation the evidence supports is that it is the size of the QDots that hinder their ability to access targets in the nucleus or large protein complexes. This explanation further highlights how little we really know about the 3D structure of protein complexes in the cell and the effect of fixation and permeabilisation upon them. Why for example, can tubulin be labelled with QDots but F-actin cannot, despite them both being abundant filamentous cytosolic structures? At this point we can’t say.

So why is this study important? Publication bias (the preferential publication of ‘positive’ results) has largely hidden the complications of using QDots for immunofluorescence. We and others have spent time and money, trying to optimise and troubleshoot experiments that upon closer study, have no chance of working. We therefore hope that by undertaking and publishing this study, other researchers can be better informed and understand when (or whether) it might be appropriate to use Quantum Dots before embarking on a project.

This paper was published in the Beilstein Journal of Nanotechnology, an Open Access, peer-reviewed journal funded entirely by the Beilstein-Institut.

UPDATE [2017-06-13]: in response to a comment below, I’ve updated the overlay figure to use green/magenta instead of green/red. The original figure can be seen in the paper or here.