2017 Sée Lab Nuffield Students

In the summer of 2017, two year 12 students from the North West of England visited the Centre for Cell Imaging on their Nuffield research placements.

Their placement involved testing a semi-high-throughput screening method for anti-cancer drugs using cell-migration as their readout. They worked with a Glioblastoma cancer cell line during their time in the facility and shared the following comments about their experiences:

 

Charlie Fogg:

“I believe that this summer placement at the University of Liverpool was the greatest experience of my life, and I will always remember it as the reason I firmly decided that this was the career in which I needed to pursue. I believe that this summer was an eye-opening experience into the real world of science, specifically cell microscopy, and it gave me countless new ideas and theories which I will take away with me into the future, and hopefully begin to research into myself one day. The placement inspired me to want to carry on pursuing science for the rest of my life and fed my ambition to achieve in a new world which I now see with many more possibilities than I had originally perceived.”

Fahda Albaba:

“This summer was not the same of all my previous summer, it was amazing and interesting because I spent it in department which I’d like before to be on, I learnt a lot of useful things: using high demand microscope, experiment skills as well as the importance of organisation, planning  and time management for each project. This placement gives me the chance to recognise the enjoyable feeling of practical and research world. Also this project allows me to deal comfortably with analysed imaging software which I am never deal with before, these wonderful software will make me think more deeply about the experiment, is not the same of the past (just follow the instructions).”

 

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U87 cells stably expressing a red nuclear marker. Timestamps are HH:MM

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Quantum dots for Immunofluorescence

Guest post by Dave Mason; reblogged from rapha-z-lab

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. 

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.