open access

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.

Francis_et_alFig3_GM

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.

2190-4286-8-125-graphical-abstract.png

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.

We are anonymous (or not). We need you to join. We are (mostly) making scientific discussion in the open possible and easy.

The last thing you probably want to read is one more article about anonymity in (post-publication) peer review. The topic has been covered recently by Bastian, Blatt, Lawrence, Oransky, Moriarty & PubPeerNeuroskeptic, Schneider to name just a few. I am sorry. I’ll keep it short.

I have decided to sign the peer review reports I write as a referee. Yet, I insist that attacks against anonymity in post-publication peer review are unfair, misguided and counterproductive. These two positions might seem contradictory. Bear with me to the end of the post and, hopefully, you might agree they are not.

Social media (and in particular PubPeer) have played a role in pretty much all recent scientific controversies, in part because the traditional channels are at best inefficient and at worst useless. Journal editors and some authors do not know how to react when criticism of articles appear on these platforms. One possible reaction is to shoot the messenger. If it is anonymous, call them anonymous cowards and question the motives. If it is not, call it cyber bullying.

Philip Moriarty, a strong supporter of PubPeer, has nevertheless titled his blog contribution to this debate “We are anonymous. We are legion. We are (mostly) harmful.” With friends like this, who needs enemies… I, and others, have responded in the comments section.

We can argue all of 2016, but the key practical question are the following. Your colleague/student has read a paper and has some interesting comments that she would like to share with the world;

Say you have chosen the first option and your colleague/student has now posted her critique on PubPeer (anonymously). You happen to also know the authors.

These are very simple questions. How we answer them has implications. Peer review is central to our practice, yet publicly engaging in scientific discussion on someone else’s work is often seen as not nice. This is the cultural barrier that we need to break. Many authors make the choice of not responding to carefully crafted criticism of their work (whether the criticism is anonymous or not). A colleague recently contacted me querying my opinion on a paper. We exchanged a few emails. We agreed there were several problems and possible errors of interpretation. I suggested to share our critique on PubPeer thereby giving the original authors the opportunity of a reply (and the rest of the community the opportunity to contribute to the discussion). He replied (SIC, smilies included): “Sorry, not for me 🙂 I do not like sharing 🙂“. This is the culture we have to change. We did not even get to discuss the possibility of anonymity.

Leonid Schneider argues that most of PubPeer is not really post publication peer review. Instead, it is calling out fraud. While anonymity is OK when you call out fraud, it would problematic in cases of scientific arguments. Even if we accepted the latter (which I do not), the distinction is artificial. There is a continuum of practices from outright fabrication to cherry picking of data and extremely optimistic interpretation of results (Twitter convo on this point here), e.g. it is common that extensive statistical analysis of data is necessary to demonstrate fraud. Any analysis of published data is post-publication peer review, whether it results in new hypotheses, questions and clarifications, or suspicions of fraud.

I disagree that anonymity is a problem and I can see plenty of valid, honest, reasonable (and even anodyne) reasons why you might choose to comment anonymously. If you insist it is a problem, fine, but I hope you will agree that it is secondary to getting valuable critiques of published work in the open. And, most importantly, do not side with Blatt who is calling for authors not to respond to criticism of their work.

It is sometimes argued that anonymity in peer review is fine because there is someone, the editor, who knows the identity of the reviewer. The power asymmetry, and therefore the potential for abuse, is however much larger in formal peer review than in post-publication peer review. At PubPeer, an anonymous comment stands purely on its merits; a scientifically strong response from the authors will bring credit to the authors. In formal peer review, the anonymous referee comes with the prestigious vetting of the editor. Furthermore, editors who are often not experts and always stretched with time, rely largely on these reports for their decisions; those have an impact on career progression, eventually grant funding, etc. The accountability of reviewers is little to none. I have started to sign my reviews during the course of 2015 with some hesitations, but my commitment is now firm. I have had some positive feedback and in one case (where I had rejected the paper), an email from an author asking me for further advice on their revised ms before submitting elsewhere. For more reasons to sign your reviews, check this.

So, to conclude, here is my advice for 2016: contribute to PubPeer (anonymously, or not, I care little), sign your peer reviews… and publish them when possible.

 

 

 

 

 

Extracting diffusion dynamics from the fluctuations in photothermal images

 Dan-z-earth

This is a guest post by Dan Nieves, who was until recently a joint member of Raphael and Dave’s labs. Dan has moved as far as he could go from us: he is now residing in Sydney at the EMBL Australia node for Single Molecule Science at the University of New South Wales.

Today, our paper from my time at Liverpool “Photothermal Raster Image Correlation Spectroscopy (PhRICS) of gold nanoparticles in solution and on live cells was published in the new Royal Society open-access journal, Royal Society Open Science.  This journal is committed to an open peer-review system, thus, the review history and referees comments are viewable alongside the article, and also post publication peer-review in the form of a comments section below the paper is facilitated. Additionally, the data that supports the conclusions of the paper are (and have to be) readily accessible (here at Figshare). This is exciting, as not only are the discussions between authors and referees are available to everyone, but you can also join in the discussion fully after publication with access to the primary data. Therefore, the critical evaluation/re-evaluation of the work is totally encouraged and should never stop!

Our paper describes the development of an extension of photothermal heterodyne imaging; a technique used to detect and image single gold nanoparticles much smaller than the diffraction limit at high signal to noise via scattering induced by laser light absorption (nice explanation here). The extension employs fast raster-scan imaging of the sample in which fluctuations, or “streaks” (top panels, Fig.1), are observed due to the movement of nanoparticles through the detection volume during the scan. From these fluctuations it is possible to extract how fast the nanoparticles are moving from the application of image correlation analyses.  In our particular case, we applied the raster image correlation spectroscopy (RICS) method, developed in the lab of Enrico Gratton (original paper here). Briefly, after acquiring many raster scan images; the images are then spatially correlated with themselves by shifting the image pixel by pixel in all directions (x and y in this case) and calculating the correlation function.  This means repeating fluctuations within the image, i.e., nanoparticle diffusion, will be reflected in the time it takes for the spatial correlation to decay, for example, the spatial correlations for movement of slow moving objects decays quite differently to that of fast moving objects (lower panels, Fig.1). From the spatial correlations the diffusion behavior, such as the diffusion coefficient, can be extracted.  In our case, I applied the analysis to photothermal images of 8.8nm gold nanoparticles diffusing in solutions of different viscosity to verify the PhRICS approach (Fig.1). Here, we were able to extract the diffusion coefficients of the nanoparticles in the different solutions. The advantage of this approach compared to the current photothermal heterodyne techniques for probing diffusion (photothermal tracking and absorption correlation spectroscopy) is that not only can we acquire rapidly information on fast diffusion dynamics, but we can also observe the distribution of nanoparticles over the relatively large area of the image (≈ 40 μm2).

 

Fig.1

Fig.1 – Example of gold nanoparticle diffusion in solutions of different viscosity (top panel) with the corresponding spatial correlations (bottom panel).

We then turned our attention to the use of the technique to observe the diffusion of fibroblast growth factors labelled with gold nanoparticles on live cells. FGFs are involved in a wide range of essential biological processes from the formation of morphogen gradients and signalling to homeostatic control of glucose and phosphate levels.  Here, 8.8 nm gold nanoparticles were used to covalently label single fibroblast growth factor 2 proteins (FGF2-NP: via this method), and then incubated with live rat mammary fibroblast cells (Fig.2).  It was observed previously in our lab that there is significant heterogeneity in FGF2 distribution and diffusion in the pericellular matrix when bound to heparan sulphate. We found the diffusion coefficient of the FGF2-NP could be extracted, and that diffusion measurements were variable depending on the area imaged.  Additionally, it is apparent that the image data contained much more information than we could extract using the simple diffusion model applied.  The observation of the formation and dissolution of intense peaks within the images, added to the 2D-movement of such peaks from image to image (see Movie1), gives more insight into the dynamic long range restructuring of the pericellular matrix of live cells at “short” (μs and ms) and “longer” (secs and mins) timescales.

Clipboard

Fig.2 – Photothermal image of rat mammary fibroblast incubated with 600 pM of FGF2-NP.  Blue boxes indicate the areas where PhRICS imaging was performed on the cell.

Cell_1_600pMFGF2_RICS_5_STACK

Movie1 – PhRICS image series from box 5 in Fig.2

The paper is now available at the Royal Society Open Science , and if your interest has been piqued thus far, I strongly encourage you check the paper out.  Better still would be for you to engage in the post-publication comments section should you have any questions, comments or suggestions.

A good day for science; respect to the Editor…

Earlier, I reported on the publication of our article on the internalisation of peptide-capped nanoparticles in cells. Today, I want to share with you the publication process as it happened at PloS One. The paper was submitted on the 20th of November 2014. The academic editor sent his decision, major revision, along with two referees reports on the 22nd of December, i.e. one month after submission [great turn around time!].

Reviewer 2 was very supportive but reviewer 1 much less so: there appeared to be a real difference of interpretation regarding the impact of cell-penetrating peptides on the intracellular localisation of ingested nanoparticles. The reviewer also requested additional experiments that we could not easily do at this time and that we felt were unnecessary to support our main conclusions. The academic editor himself, Dr Pedro V. Baptista [more on PloS One editorial process here], was author on a paper which in some ways could be seen as conflicting with our results and interpretation. The response to the referees and editors took me a long time to write. It was submitted on the 29th of January. I share it below.

The paper was accepted on the 6th of February. I welcome this decision, not just because our paper gets published -this is of course also great news!-, but because it demonstrates that there is space for open scientific debate in the peer reviewed literature. For this, I am immensely grateful to Dr Baptista.


Response to the referees.

Dear Dr Pedro V. Baptista

On behalf of my co-authors, I would like to thank you for handling our article and to thank the reviewers for their careful reading and for their comments.

Reviewer 2 notes that the context of our ms is the existence of conflicting reports on the effect of TAT and HA2 on intracellular fate of nanoparticles. Indeed, some articles have reported efficient access to the cytosol, while other studies indicate that most particles remain confined in endosomal compartments. Our own experiments are in line with this second group of articles. Reviewer 2 notes that “the study is well designed and executed and the results are interpreted appropriately”. Reviewer 2 supports publication in its current form.

Reviewer 1 has concerns about novelty. Reviewer 1 also suggests that we should add three references. These fall in the first category mentioned above, i.e. articles that support the notion that TAT enables access to the cytosol. It is of course appropriate that we should cite studies from both groups of articles. One of the three, […], was in fact already cited. We have now added the other two, i.e.: […]

Experiments related to this topic have led to many articles in the past 10 years. However, the persistence of conflicting reports and the importance of the topic for many envisioned applications require new insights. This we have provided through the use of imaging modalities that provide information across different scales: electron microscopy measures what occurs to a few nanoparticles in a very small part of the cell; photothermal microscopy measures what happens to the bulk of nanoparticles across a large part of the cell. This combination is thus uniquely able to address, in at least one cell type and a particular formulation of nanoparticle, the fate of TAT-functionalised nanoparticles after they bind to the cell surface.

Below we respond to the detailed queries of reviewer 1 and trust that the manuscript now meets the standards required for publication in PLOS One.

Dr Raphaël Lévy, rapha@liverpool.ac.uk

Detailed response to reviewer 1 queries:
• Novelty. Our article is a significant piece of work that adds useful information towards understanding and clarifying the impact of cell penetrating peptides on intracellular localisation of nanoparticles. The work is novel because it builds on a new imaging methodology that directly images the nanoparticle cores (as opposed to an attached fluorescent molecule) and gives a better overview of an entire cell than just electron microscopy. It is also novel because our peptide self-assembled monolayer approach enables us to do systematic variations of the surface chemistry of the nanoconjugates.
• “To include as a new figure, the extinction spectra of all the nanoconjugates as well as all the scattering spectra […]”. The reviewer is right that extinction spectra are very useful to characterise functionalisation and colloidal stability. We have added the requested figure as Fig. S0. For the conjugates used in Fig. 1, the formation of the self-assembled monolayers results in a minimal shift of the plasmon band of ~1-3 nm. This shift is small compared to the width of the plasmon peak. Because photothermal microscopy relies on absorption at the wavelength of our heating laser which matches the position of the maximal absorbance, differences due to a 1-3 nm plasmon shift are negligible. Interestingly, particles presenting a higher percentage of TAT in their monolayer do show a larger plasmon shift indicative of aggregation. We have modified the paragraph on the formation of the SAMs as follows: “Formation of the monolayer was immediately visible because of the increased colloidal stability and of a small red shift of the nanoparticles plasmon band (Fig. S0). Higher proportions of TAT in the monolayer resulted in nanoparticle aggregation and therefore were not used for further studies (Fig. S0).”

• “To include the images and quantification in Figure 1 with cells only with naked gold nanoparticles and cells only with PEG-gold nanoparticles and compare intensities.” The images and quantification for “cells only” were already included (Fig. 1A and first column of Fig. 1F). We have not included “naked gold”. Instead, as a reference point, we have used PEG-gold particles that have a capping composition made of CALNN and CCALNN-PEG. “naked gold” does not remain naked: non-specific adsorption of proteins, e.g. serum albumin in the cell medium, very rapidly changes the properties of the surface [see for example, Time Evolution of the Nanoparticle Protein Corona, Casals et al., ACS Nano, 2010, 4, pp 3623–3632]. The CALNN and CCALNNPEG composition was optimised, as discussed p 7, line 213-220 and Fig. S2 “Gold nanoparticles uptake decreases with increasing percentages of CCALNN-PEG”. The selected composition leads to minimal uptake as shown in Fig. 1B and the second column in Fig. 1F. From this reference composition, we have made systematic variations where we include defined percentages of the two functional peptides (dHA2 and TAT). For all these conditions, exemplary images are shown in Fig. 1 A-E, additional images are shared via figshare (http://dx.doi.org/10.6084/m9.figshare.1088379) and the quantifications are shown in Fig. 1F.

• “To perform other technique to quantify the gold content […].To include more time points in the TEM studies […]. […] the efficacy results reported by the authors are premature without the additional data described above.” While we agree that the suggested experiments are interesting, they are not necessary to reach the conclusions arrived at in the ms. Those conclusions do not concern “efficacy”, but increased uptake and intracellular localisation. The increase in photothermal signal as well as in the counts of nanoparticles in EM images unambiguously demonstrate increased uptake. The non-homogenous distribution of signal observed in the photothermal images and the electron microscopy analyses unambiguously rule out cytosolic distribution of the nanoparticles. The time point of 3 hours used in our studies is a key point both from the perspective of applications and of cell entry mechanisms. We agree that a systematic analysis as a function of time after uptake would provide further insights into endocytotic mechanisms, but it is outside of the focus of this study. Furthermore, it has been done extensively by cell biologists since the 1950s using a variety of probes. Notably, one of the first applications of gold nanoparticles in biology precisely focused on the mechanisms by which cells probe their external environment (Electron microscopy of HeLa cells after ingestion of colloidal gold, Harford et al., J Biophys Biochem Cytol 1957 3:749-756; reference added into the ms).

The standards in the field have been to publish only one or two representative electron
microscopy images. The photothermal imaging provides a unique means for the reader to understand nanoparticle distribution over biologically representative scales. Importantly, we are sharing here 942 EM images and 37 photothermal images. By publishing all of our data alongside the study [1], we enable other scientists to check and challenge our conclusions and propose alternative hypotheses. PLoS One is a particularly good forum for our article because of its commenting platform where this discussion can continue in the open after the publication of the article.
[1]. DOIs of the data:

10.6084/m9.figshare.1088379, 10.6084/m9.figshare.875584, 10.6084/m9.figshare.875630, 10.6084/m9.figshare.875545, 10.6084/m9.figshare.875477, 10.6084/m9.figshare.874219, 10.6084/m9.figshare.874153, 10.6084/m9.figshare.874033, 10.6084/m9.figshare.873852, 10.6084/m9.figshare.1088399, 10.6084/m9.figshare.1246458, 10.6084/m9.figshare.1246609,
10.6084/m9.figshare.1246622, 10.6084/m9.figshare.1246660, 10.6084/m9.figshare.1246696, 10.6084/m9.figshare.1246707

Novelty, reproducibility, and data sharing in (nano)materials science

Half-random ranty post that might develop into something more structured at some point… Feedback very much welcome.

Andrew Maynard has blogged about the extent to which novelty should (or, in fact, should not) be the main consideration for the evaluation of nanomaterials risks (initially published as an editorial in Nature Nanotechnology). It’s entitled “Is novelty in nanomaterials overrated when it comes to risks” and is well worth reading in full. A central point is that:

Novelty as a result is a subjective, transient, and consequently a rather unreliable indicator of potential risk. It tends to obscure the reality that conventional behaviour can sometimes lead to harm, and that mundane risks are still risks. And it favours the interesting (and possibly the headline-grabbing) over the important. But if novelty is an unreliable guide to potential risk, how can approaches be developed that help identify, understand and manage plausible risks associated with emerging materials and the products that use them?

Apparently unrelated (but wait for the next paragraphs), there are various initiatives to encourage or even mandate sharing of data related to the characterization of (nano)materials. It is thought that this will boost innovation and facilitate the coming together of computational and experimental work. Maybe the most impressive and concerted effort comes from the White House Office for Science and Technology as exemplified by this post It’s Time to Open Materials Science Data. Publishers have smelled something and are moving to the area of providing services for data sharing and curation; NPG launched Scientific Data in partnership with FigShare; Elsevier has just launched an initiative specifically targeted to open data in Materials Science.

Now for the (arguably subtle and tenuous) link. Novelty is overrated not just when it comes to risk. It is overrated in materials science full stop. This seems not intuitive; surely scientific endeavour in materials science is about discovering new materials. The problem here (and arguably the opportunity too) is that there is an immense combinatorial space of potential new materials. We work on peptide-capped gold nanoparticles. By varying the peptide sequences and making various mixed monolayers, we can potentially generate hundreds of novel materials every day (and we do make a fair number). The combinatorial space of potential nanomaterials vastly exceed the number of potential molecules. Most of these materials are not interesting, but they are novel: nobody made them before.

I see a lot of research articles which can be summarised as

  1. This is a novel nanomaterial (and it truly is: nobody has made before this gold-nanorod-with-carbon-dots-at-the-tips-graphene-oxide-on-the-side-and-some-antibody-labelled-conductive-polymer-wrapped-around [1])
  2. It could be used for [delete as appropriate] energy/biological imaging/curing cancer (and it will never be).

When it comes to safety, Andrew argues convincingly that the focus should be on plausible scenarios rather than on novelty. When it comes to what should be curiosity-driven science, there seems to be a lot of new materials generated for the sole purpose of highly improbable applications rather than in the pursuit of general principles that would help us explore the materials landscape. This has the very unfortunate consequence that the materials characterisation is often poor and limited to whatever is thought to enable the envisioned application. An extremely large proportion of these new materials are made by a single group for the purpose of a single paper. The experiments are not reproduced independently. Capturing all of this data into platforms that are open and suitable for data mining is a noble and worthwhile purpose which I support, but it must be accompanied by a change of focus and higher standards of characterisation otherwise I fear that it will not help understanding much.

Thanks to who chronicled the reaction of materials scientists to an OFST presentation at the MRS conference in Boston in December 2014.

[1] Novel Nano-Lychees for Theranostics of Cancer; Charles Spencer and Edna Purviance; Nature Matters-to-all (2015) 7  101-114