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.

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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.

Lettre d’Angleterre: Souveraineté

English version below.

Le Front National veut soi-disant redonner à la France sa souveraineté en la sortant de l’Union Européenne. Aux oreilles d’un résidant en Angleterre, ce discours est familier : c’est celui, répété ad nauseam par Leave.eu et les supporters du Brexit : reprenons le contrôle de nos lois et de nos frontières (‘Take back control’). C’est aussi une illusion. Pour deux raisons.

La première raison est que le parlement du Royaume-Uni était souverain. Ce n’est pas moi qui le dis mais le livre blanc produit par le gouvernement de Theresa May lui-même : « La souveraineté du Parlement est un principe fondamental du Royaume-Uni. Bien que le Parlement soit resté souverain durant toute la période où nous avons été membre de l’Union Européenne, on n’en pas toujours eu le sentiment. »

La deuxième raison, peut-être contre-intuitive, est que, au-delà de la souveraineté du processus législatif, la capacité du Royaume-Uni à prendre en main son destin de manière indépendante est mise à mal de manière spectaculaire par le Brexit. Son gouvernement, isolé, sera encore moins capable de résister aux lobbys, comme l’explique admirablement… Rupert Murdoch répondant à la question pourquoi êtes-vous tellement opposé à l’Union Européenne : « c’est facile, quand je vais au 10 Downing Street, ils font ce que je leur demande, quand je vais à Bruxelles, ils m’ignorent complètement. »  On voit mal un Royaume-Uni isolé faire face à Microsoft, Google ou Apple. De plus, quittant le marché commun, le Royaume-Uni doit négocier des accords bilatéraux avec d’autres pays, et, ces pays, vont imposer leurs conditions, d’autant plus qu’il est clair que le Royaume-Uni est en position de faiblesse. Theresa May a choisi d’aller en Inde pour l’un de ses premiers voyages après le referendum. L’Inde insiste que tout accord économique devra s’accompagner d’un assouplissement des règles d’immigration et de visa. Theresa May devra donc choisir entre le contrôle de l’immigration (‘Take back control’) et un accord commercial avec l’Inde. Chaque pays (y compris ceux de l’Union Européenne) aura ses propres demandes auxquelles le Royaume-Uni devra bien souvent se soumettre.

Le Brexit est un abandon de souveraineté.

*********

The National Front is pretending that it will give France its sovereignty back from Brussels. To my ears, this is familiar discourse: it is the one peddled ad nauseam by Leave.eu and other Brexit supporters: Take back control (of laws, borders). It is an illusion. For two reasons.

The first reason is that the UK Parliament was sovereign. Not my word, but the white paper produced by the government of Theresa May herself: “The sovereignty of Parliament is a fundamental principle of the UK constitution. Whilst Parliament has remained sovereign throughout our membership of the EU, it has not always felt like that.

The second reason, maybe counter-intuitive, is that, beyond the sovereignty of the legislative process, the UK capacity to shape its own destiny is badly damaged by Brexit. Its government, isolated, will be even less able to resist pressures from lobbies, as explained transparently by…  Rupert Murdoch, who, replying to the question of why he was so opposed to the European Union, replied. ”That’s easy, when I go into Downing Street they do what I say; when I go to Brussels they take no notice.’ It is hard to imagine the UK, isolated, courageously facing Microsoft, Google ou Apple. Furthermore, leaving the common market, the UK must negotiate new bilateral agreement with third countries, and, these countries will impose their conditions, especially since it is clear the UK is in a weak position. Theresa May chose to go to India for one of her first travel after the referendum. India insists that any agreement will need to include a softening of immigration and visa rules. Theresa May will therefore need to choose between control of immigration (‘Take back control’) and a commercial agreement with India. Each country (including those of the EU will have their own demands to which the UK will often need to submit.

Brexit is a loss of sovereignty.

Lettre d’Angleterre

J’ai cru que la doctrine et l’histoire d’un peuple si extraordinaire méritaient la curiosité d’un homme raisonnable.

Voltaire, 1734, Lettre d’Angleterre.

Je ne suis certes pas Voltaire, mais, peut-être puis-je prétendre être un homme raisonnable?

J’habite Liverpool depuis 15 ans. Les événements politiques en Angleterre durant les 12 derniers mois sont extraordinaire, et, particulièrement important à considérer à trois jours du premier tour de l’élection présidentielle française.

Ce qui se passe ici, c’est une dislocation de la société, travaillée par les mensonges et la haine. Avec la légitimation toujours plus grande d’un discours qui fait appel à « la volonté du peuple » pour « écraser les saboteurs » . Un discours violent qui rejette toute discussion rationnelle et informée, au profit d’un tribalisme viscéral, émotionnel et souvent xénophobe.

C’est pourquoi je tremble lorsque j’entends ce même appel au peuple, « le gros animal, un monstre qui ne connaît ni la vertu ni la raison » (Philippe Val/Socrate) chez deux candidats qui peuvent se qualifier pour le second tour (Au-Nom-Du-Peuple-LePen et La-Force-du-Peuple-Mélenchon): je suis affolé.

Ces deux candidats qui ont des amitiés particulières pour divers dictateurs.

Ces deux candidats qui partagent la volonté explicite ou implicite de détruire l’Europe et ses institutions. L’Europe qui a contribué énormément à la prospérité retrouvée de cette belle ville de Liverpool et de beaucoup d’autres régions défavorisées sur le continent. L’Europe qui a garanti la paix pendant les derniers 70 ans (un ex-dirigeant du parti conservateur a proféré des menaces de guerre contre l’Espagne à propos de Gibraltar. En Avril 2017). L’Europe qui, seule, nous donne une voix suffisamment forte pour défendre nos principes démocratiques, parler d’égal à égal avec les autres grandes puissances, qu’elles soient des états ou des multinationales, et, affronter le défi majeur du changement climatique.

L’enjeu ne pourrait être plus grand.

SmartFlare controversy: independent confirmation of endosomal localization

Check this previous post for a quick summary of the SmartFlare controversy, or read all SmartFlare-related posts if you are really passionate.

At the centre of the SmartFlare controversy is the rather simple question, from an experimental point of view, of how many Spherical Nucleic Acids (to use Chad Mirkin’s terminology), if any, escape the endosomal pathway.

In contradiction with Chad Mirkin’s many peer reviewed articles and EMD Millipore marketing material, we concluded (Mason et al, 2016) that the Spherical Nucleic Acids do not escape endosomes and do not detect cytosolic mRNAs.

A few days ago, Sven Budik et al, an Austrian group published their evaluation of the SmartFlare in the context of equine embryo development. They write: “In all positive cells,
regardless of whether they occurred in equine conceptus, trophoblastic vesicle or fibroblast cell culture, the fluorescence signal showed a spotted pattern that is in accordance with the observations of Mason et al. (2016).

They also used electron microscopy to look at the intracellular localization of the particles. Here is the relevant part of their discussion and conclusion (emphasis mine):

The present study indicates that the intracellular process of nanogold particle uptake is endocytic and endosomal with a lysosomal sorting after longer incubation periods. This finding is in agreement with results from HeLa cells in vitro (Gilleron et al. 2013). Similarly, nanoparticles injected intravenously were taken up by endocytosis and later
clustered in lysosomes primarily in macrophages (Sadauskas et al. 2007). The incorporation time of lipid nanoparticlecontaining short interfering RNA gold particles in HeLa cells was similar (Gilleron et al. 2013) to that demonstrated in equine trophoblast vesicles in the present study. Accumulation of SmartFlare probes in residual bodies may be a consequence of increased stability of the immobilised oligonucleotides adjacent to the nanogold particles due to enhanced nuclease resistance (Rosi et al. 2006). In accordance with the results of Mason et al. (2016), we observed no or very few nanogold particles free in the cytoplasm, confirming a primarily endosomal and lysosomal localisation.

This observation raises the question how a specific SmartFlare probe is able to detect its target mRNA located in the cytoplasm. One possible explanation for the generation of lysosome-located specific fluorescence signals by SmartFlare probes could be the existence of specific RNA sequences imported for subsequent degradation into lysosomes (Fujiwara et al. 2013). Further studies using qRT-PCR investigating the isolated lysosomal fraction before and after incubation with specific SmartFlare probes are necessary to confirm this hypothesis. An 18S RNA nano-flare probe had a dose-dependent cytotoxic effect on porcine fetal fibroblasts (Fu et al. 2016). In contrast, no cytotoxic effects or changes in morphology after incorporation of antisense oligonucleotide nanogold particles in a mouse endothelial cell line were observed by Rosi et al. (2006). In addition, in the present study, there was no evidence that incubation with the SmartFlare probes had a toxic effect on the equine cells tested, even at higher concentrations. This is in accordance with the results of Pan et al. (2009) demonstrating that 15-nm gold particles have only low cytotoxic effects compared with the detrimental effects of small 1.4-nm gold particles.

In conclusion, SmartFlare probes pass into early equine conceptuses at stages used for embryo transfer, as well as trophoblast vesicles and cells cultured in vitro. In these early ZP equine conceptuses, the time frame (.5 and ,24 h) for SmartFlare uptake would be suitable for practical applications in commercial embryo transfer programs. Therefore, these probes are suggested to be applicable to pre-implantation genetic diagnosis before transfer of these conceptuses to the recipient.

In summary, the authors’ results are entirely consistent with our observations. They conclude, quite reasonably, that if SmartFlares detect mRNAs whilst being in endosomes, they cannot directly detect cytosolic mRNAs. This is in direct contradiction with Mirkin et al and EMD Millipore. Then, they propose that if the SmartFlares work, they maybe detect mRNAs which are in endosomes. This an interesting hypothesis that will require further study and is very different from anything published by Mirkin and EMD Millipore (the relevant reference is here). Since Budik et al do not provide any evidence that the SmartFlares actually detect mRNAs in the first place, maybe a simpler explanation is that the SmartFlares signal is unspecific and result from the probe degradation by nucleases in endosomes.

Thoughts on #LiveTweeting

Dave Mason on why you should be live-tweeting at conferences

Blog and Log

As a part of the Centre for Cell Imaging and a member of the Microscopy and BioImage Analysis community, I occasionally get away to conferences like the recent NEUBIAS training school and symposium in Portugal.

vessels_neubias_banner

Since having joined Twitter last year (@dn_mason), this is the second conference that I’ve been to, and as a result, was the second time I tried (with reasonable success) to Live Tweet at the conference.

Live What Now?

Going right back to basics, Twitter is a platform for broadcasting small messages (of ~140 characters). Some describe it as micro-blogging. To many, the brevity of each tweet is both it’s greatest strength and also one of the most frustrating features.

Live tweeting, is basically the act of providing a running commentary of a seminar, event or even a whole conference. All of the tweets associated with such an event can be tied together using…

View original post 1,117 more words

Publication bias. Grant bias.

All academics writing grants will tell you this: if you want to be successful when applying to a thematic research grant call, you must tick all of the boxes.

Now, imagine that you are a physicist, expert in quantum mechanics. A major funding opportunity arises, exactly matching your interest and track record. That is great news. Obviously you will apply. One difficulty however is that, amongst other things, the call specifies that your project should lead to the “development of highly sensitive approaches enabling the simultaneous determination of the exact position and momentum of a particle“.

At that point, you have three options. The first one is to write a super sexy proposal that somehow ignores the Heisenberg principle. The second option is to write a proposal that addresses the other priorities, but fudges around that particular specification, maybe even alluding to the Heisenberg principle. The third option is to renounce.

The first option is dishonest. The second option is more honest, but, in effect, is not so different from the third: your project is unlikely to get funded if you do not stick to the requirements of the call, as noted above. The third option demonstrates integrity but won’t help you with your career, nor, more importantly with doing any research at all.

And so, you have it. Thematic grant calls that ask for impossible achievements, nourished by publication bias and hype, further contribute to distortion of science.

OK, I’ll confess: I have had a major grant rejected. It was a beautiful EU project (whether BREXIT is partly to blame I do not know). It was not about quantum mechanics but about cell tracking. The call asked for simultaneous “detection of single cells and cell morphologies” and “non-invasive whole body monitoring (magnetic, optical) in large animals” which is just about as impossible as breaking the Heisenberg principle, albeit for less fundamental reasons. We went for option 2. We had a super strong team.

How to Characterize Gold Nanoparticles’ Surface?

Guest post by Elena Colangelo

Our Topical Review on the characterization of gold nanoparticles (GNPs) has just been published in the Bionconjugate Chemistry Special Issue “Interfacing Inorganic Nanoparticles with Biology”.

The literature is abounding in works on GNPs for applications in biology, catalysis and sensing, among others. GNPs’ appeal resides in their optical properties, together with the well-developed methods of synthesis available and the possibility of functionalizing their surface with small molecules of interest, which can readily self-assemble on the GNPs’ surface forming a monolayer.

However, allegedly the structure and organization of self-assembled monolayers (SAMs) at the GNPs’ surface are in fact aspects too often neglected [though surely not on this blog; RL]. Such elucidation is challenging experimentally, but it is crucial not only to ensure reproducibility, but also to design nanosystems with defined (bio)physicochemical and structural properties, which could then be envisioned to assemble in more complex systems from a “bottom-up” approach.

Our Topical Review gives an overview of the current knowledge and methods available to characterize the GNPs’ surface with different molecular details.

capture

Cartoon illustrating the different levels of GNPs’ surface characterization discussed in the Topical Review.

First, the experimental methods commonly used to provide the basic characterization of functionalized GNPs, such as identification and quantification of the ligands within the monolayer, are detailed with the aid of some examples.

Second, the experimental methods providing information on the monolayer thickness and compactness are reviewed.

Third, considering that the SAM’s thickness and compactness do not only depend on the amount of ligands within the monolayer, but also on their conformation, the experimental methods that can provide such insights are recapitulated. However, we also stressed on the limitations intrinsic to these methods and on the challenges associated to the determination of the structure of SAMs on GNPs.

Fourth, we summarized some of the approaches used to give insights into the organization of different ligands within a SAM. Indeed, mixed SAMs on GNPs are useful since they can impart to the nanoparticles different functionalities and offer a way to tune their stability.

Fifth, highlighting again the limited insights into the SAM’s structure and organization that can be gathered with experimental techniques, we detailed some examples where a combination of experimental and computational approaches was able to provide a compelling description of the system and to assess molecular details that could not have been revealed experimentally.

Overall, this Topical Review gives emphasis on the importance of GNPs’ surface characterization and on fact that even though a number of experimental techniques are available, they are intrinsically limited and they cannot provide a fully detailed picture. Hence, it is advantageous to combine experimental and theoretical approaches to design nanoparticles with desired (bio)physicochemical properties [such as, e.g., our paper under review, currently available as a preprint; RL].