David Mason

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.

How smart are SmartFlares?

This post is co-authored by Raphaël Lévy and Dave Mason.

Note: We contacted Chad Mirkin and EMD Millipore for comments. Chad Mirkin replied but did not allow me to share his comments as he prefers to discuss his work in peer reviewed manuscripts rather than blogs. EMD Millipore has provided a response (reproduced below) and is keen to further engage in the discussion.  They wrote that they “look forward to responding to [the questions you pose at the end of the post] after your blog is posted so other researchers who may have the same questions can follow our discussion online.” [Update: they have gone into silent mode after that comment].

Update: The results of our study have now been published.


To image proteins in cells, biologists have powerful tools based on the Green Fluorescent Protein (GFP) for which Osamu Shimomura, Martin Chalfie and Roger Y. Tsien  obtained the 2008 Nobel Prize in Chemistry. RNA molecules play crucial roles in cells such as coding, decoding, regulation, and expression of genes, yet they are much more difficult to study. SmartFlares are nanoparticle-based probes for the detection and imaging of RNA in live cells. Could they become the GFP of the RNA world?

Many certainly believe this to be the case. SmartFlare ranked second in TheScientist top ten 2013 innovations, with one of the judges, Kevin Lustig, commenting “These new RNA detection probes can be used to visualize RNA expression in live cells at the single-cell level.”  The following year, SmartFlare won an R&D100 award. The technology comes from Chad Mirkin’s lab at Northwestern University. Chad Mirkin is the winner of numerous prestigious prizes and a science adviser to the President of the United States. The scientific articles introducing the SmartFlare concept (under the name of Nano-Flare) were published in the Journal of the American Chemical Society in 2007, ACS Nano in 2009, etc. In 2013, the SmartFlare technology was licensed to EMD Millipore. Here is one of their promotional video:

For a molecular sensor to work, it needs a detection mechanism. The principle of the SmartFlare is explained from 0:45. A capture oligonucleotide (i.e. DNA) is bound to the gold nanoparticles. A reporter strand is bound to the capture strand. The reporter strand carries a fluorophore but that fluorophore does not emit light because it is too close to the gold (the fluorescence is “quenched”). In the presence of the target RNA, the reporter strand is replaced by the target RNA and therefore released, quenching stops, and fluorescence is detected. The release is shown at 2:05. Simple and convincing. Gold nanoparticles are indeed excellent fluorescence quenchers (we have used this property in a couple of papers).

But, for a molecular sensor to work, it also needs to reach the molecule it is supposed to detect. The SmartFlares are shown at 1:40 entering the cells via endocytosis, a normal mechanism by which the cell engulfs extracellular material by entrapping them into a bag made of cell membrane. Molecules and particles which enter the cell by endocytosis normally remain trapped in this bag. This entrapping is essential to protect us from viruses and bacteria by preventing them from accessing the cell machinery. Here, however, at 1:45 – 1:46, something truly remarkable happens: the endosome (the bag) suddenly fades away leaving the particles free to diffuse in the cell and meet their RNA targets. This is a promotional video so you might say that the demonstration of, and explanation for, this remarkable endosomal escape is to be found in the primary literature but that is not the case.

SmartFlares_scheme

There is an extensive body of literature (not related to SmartFlare) dealing with endosomal escape. Some bacteria (like Listeria which can cause food poisoning) and viruses (like Influenza or HIV) use proteins to destabilise the endosome, escape and cause disease. Other mechanisms involve altering the ion balance in the endosome to pop it like an over-inflated balloon (you can read more about the ‘Proton Sponge Effect’ in this review). The problem is that none of these scenarios are applicable to gold nanoparticles conjugated to oligonucleotides. The problem is compounded by the choice of techniques used to analyse SmartFlare uptake into cells. Most of the published papers (for examples see here, here and here) characterise “uptake” and do so largely via Flow Cytometry or Mass Spectrometry (to measure the gold content of the cells). These papers certainly support NanoFlares being taken up into endosomes, but don’t offer any evidence for endosomal escape. A systematic unbiased electron microscopy study would enable to gather an estimate of how many nanoparticles have escaped the endosomes. Alternatively, fluorescence microscopy can be used to visualise a diffuse (released) instead of punctate (still in endosomes) distribution of intensity. While there are some images of cells having taken up NanoFlares, the sort of resolution required to discern distribution is not afforded by publication-size figures.

Wouldn’t it be nice if we had access to the original data? Researchers are often left squinting at published figures and all too often have to rely on the author’s interpretation of the data. One solution to this problem is to make supporting data available after publication. This is the idea behind the JCB Dataviewer; allowing authors to upload the original data to support papers published in the Journal of Cell Biology. The other option is to make the data available before publication, in what is called Open Research. This has the huge advantage of opening up a discussion about data, its interpretation and meaning before going through the formal peer-review process.

It is this latter technique that we are currently using to share our study of the use of NanoFlares as VEGF RNA reporters in cells. Our Open Science Notebook gives an overview of the experimental design, results and discussion, while our OMERO server is being used to host all of the original data for anyone to access. The project is still in progress, however our main findings so far are that:

In all conditions where fluorescence is seen, the distribution is consistently punctate (see all of the data here ).

So far these findings have left us with several questions, the most interesting of which are:

  1. Why do we see punctate fluorescence with the VEGF SmartFlares? If the SmartFlares are still in endosomes, they shouldn’t be able to interact with mRNA and thus fluorescence should be quenched.
  2. Why do we see signal at all in the scrambled control?
  3. Why do different cells take up varying amounts of SmartFlares? Fluid phase dextran shows approximately equal uptake in all cells.

We’re presently investigating these and other questions. As we find out more, we will continue to post the data and update the blog.


RESPONSE from EMD Millipore:

In their response, EMD Millipore pointed to a number of relevant publications suggesting that we should revise the post after having considered this evidence. We had already seen those references and we have not altered the post, but we reproduce EMD Millipore’s response below:

 Oligo-modified nanoparticle internalization and endosomal release:

·         Oligonucleotide modified nanostructures are taken in through an endocytotic mechanism.  http://www.pnas.org/content/110/19/7625.long

·         These highly anionic structures attract a counterbalancing salt cloud.  http://pubs.acs.org/doi/pdf/10.1021/jp205583j

·         This is thought to be the mechanism of release from endosomes (via osmotic pressure) 

 Observation of punctate fluorescence:

·         At short time points, when these structures are indeed in the endosomes,  or at low detection gains on a microscope (where you are adjusting for the brightest points) the staining appears punctate.  (For example- the light in a room comes from the bulb, which is the brightest, but the room is still lit.  Keeping only the brightest point in a picture would only show you the bulb.)

·         Therefore, with regards to the experiment you’ve already performed, our first suggestion would be to turn up the gain to see cytoplasmic fluorescence.

·         Here for example are some pictures showing nice cytoplasmic stain  http://www.pnas.org/content/109/30/11975/F1.expansion.html

 

Also may be of interest: 

http://www.nature.com/mt/journal/v22/n6/full/mt201430a.html

It may be worth noting some of our more recent examples of SmartFlare in the literature, spanning across cancer and stem cell research on a variety of detection platforms (flow & microscopy).  Here the punctate fluorescence is also observed, but you can also see nice cytoplasmic staining.

·         Seftor et al. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026856/

 

·         Mehta et al.  http://www.ncbi.nlm.nih.gov/pubmed/24623279