nanotechnology

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.

Nano talk for 15 years old

About two weeks ago, the Institute received an inquiry from Shevington High School near Wigan (~ 30 min drive from Liverpool). Clare Ingham, a student teacher in the School, wrote:

Recently I’ve been discussing nanoscience and nanotechnology with my year 10 students. They were very interested and enthused on the work that is being led in the north west in this area. 

Indeed, you can see on the School news blog that they really enjoyed making some model fullerenes some weeks ago [scroll down on that page to “Year 10 Chemistry”]. Clare continued:

I’d like to keep that enthusiasm high by hopefully inviting one of your research team to the school to give a short talk/Q&A with a small number of pupils in the near future on some Liverpool led developments in nanotechnology? Would this be a possibility? I’m keen to enthuse the pupils of the science they could be part of and leading in the future.

I volunteered. I have done some outreach talks before, e.g. Christmas lecture and Scibar, but I thought I’d ask Twitter for new ideas. Thank you to @Zen_of_Science@bardmital@drheaddamage@DaveFernig@PSBROOKES and @medickinson for their suggestions which I have collected in this Spotify (send me more ideas via twitter and I’ll add them there).

I visited this morning.

I was introduced as a “distinguished scientist”. I asked the children if I looked like a “distinguished scientist”. One was brave enough to say “no”. I told them a little bit my study and career path. Then, I presented to them some real “distinguished scientist” via this picture of three members of my group at a conference in France. The aim was to challenge their (?) preconceptions about scientists and make it clear that scientists look very much like them. That was not so much inspired by the Twitter response but more by my former student Rachel Gilbert’s project, the excellentThis is what a scientist looks likeas well as what I learnt through my involvement in the Institute Athena Swan committee.

We moved to “nanotechnology”. I asked them what “nano” meant. They replied:

Very small. So small you can’t see

That was a good start but of course everything is relative; 1 meter is very small compared to the Earth-Sun distance. We need to be more precise. How small is very small? From meter to millimeter, from millimeter to micrometer and finally from micrometer to nanometer. Next, I asked them for things which have dimensions in this range. Their responses, after a bit of prompting, included:

red blood cells, viruses, fullerenes and atoms

A really good base for discussion. Red blood cells a bit big for nano? Atoms, a bit small? Viruses and fullerenes: spot on! I added a few biological ingredients: proteins, DNA, membranes. I then remarked that there were two types of objects in our list. One student did get the hint and said “Biological versus non-biological” which led me to introduce how we can make things on the nanoscale via either top-down (carving a block of matter) or bottom-up (assembling parts, or better, self-assembly although I did not really get into that). Nanotechnology is in their everyday life. It is even in their pockets. I showed them this picture of how a state-of-the-art computer looked like when my parents were born. It filled a room and was infinitely less powerful than their mobile phone. I also showed them pictures of a modern transistor and of the kind of gigantic plants which are required to make these.

I asked them what they knew about light.

travel in a straight line.

speed of light is 300 ooo km/s.

white light can be separated into different colors.

I pushed a bit more on the differences between colors and a student mentioned “wavelength” but they could not really explain what this was nor how small/big were the wavelengths of visible light. Visible light is nano (blue ~ 400 nm, green ~ 520 nm, red ~ 600 nm). Nano is everywhere 😉

Since we are scientists, we do experiments. I asked for two volunteers. Before I could say one more word, I had plenty of hands up. I then specified that I required a sample from those volunteers (at that points, I think there was a hint of worry in the teacher’s eyes) but I quickly explained that I required only one hair from each (the worry dissipated). We did the hair experiment with the laser pointer as suggested by @drheaddamage (check his videos here and here). Before doing the experiment, we tried to predict the result. Given their everyday experience of light and the fact that they learnt that light travel in a straight line, the prediction is what we should see a shadow of the hair in the laser spot. The reality is quite different. We see a scattering line perpendicular to the hair with maxima and minima along the line. This is due to the fact that light is a wave. I used this experiment to show one way by which we can get information on the size of things we can’t see.

Gold nanoparticles in water

Gold nanoparticles in water. Looks like Ribena.

 

We then moved to nanoparticles, first gold, and then superparamagnetic iron oxide nanoparticles.

The superparamagnetic nanoparticles are also quite fascinating as you can move the liquid around with a magnet and even defy gravity (picture below).

To conclude my presentation and link with our current research efforts, I explained the need to track STEM cells in the body and how those nanoparticles, both the superparamagnetic iron oxide nanoparticles and the gold nanorods, can be developed as contrast agents for animal/human imaging.

spions-small

Superparamagnetic nanoparticles in solvent. The liquid is held up by the magnet.

 

Update: Proof that I have been there; from Shevington High School news: