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.

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.

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.

 

The story behind the paper

We recently published a paper on genomic surveillance of a diarrhoeal pathogen Shigella sonnei across Latin America which represented the culmination of over five years of collaboration, as well as training and development in the region

In collaboration with the Wellcome Trust Sanger Institute, the Pan American Health Organisation and PulseNet Latin America and Caribbean (PNLAC), we whole genome sequenced over 400 Shigella sonnei collected from nine countries over two decades. Shigella are the most important bacterial cause of moderate-to-severe childhood diarrhoeal disease in low to middle income nations, and countries in Latin America still experience endemic disease and explosive outbreaks. By sharing information on common pathogen subtypes through public health networks, like PNLAC, pathogens can be traced epidemiologically to facilitate early identification and intervention in disease outbreaks. Whole genome sequencing is transforming surveillance of bacterial pathogens, as it provides the highest resolution of pathogens subtypes and can also be used to explore other genetic factors of interest, like antimicrobial resistance. However, its cost precludes routine use in some areas, which are unfortunately some of those regions where the most Shigella disease is seen.

In this study, we sequenced approximately 50 isolates from nine countries in Latin America and use whole genome phylogenetics to reveal those sublineages that were responsible for most of the disease in the region. We identified a novel global lineage of Shigella sonnei, and by correlating the geography of where isolates came from to their evolutionary relationships, we could see international transmission of some sublineages and what the distribution of different sublineages was across the continent. Visit the microreact page to play with the data yourself.

We were also able to identify key determinants of antimicrobial resistance in the pathogens and how they were distributed among the different sublineages, providing key information for managing this important disease in the region.

In addition to constructing this invaluable regional framework for ongoing surveillance, this project helped build capacity for whole genome sequencing surveillance in the region. Over the course of the collaboration, the World Health Organisation sponsored the establishment of whole genome sequencing facility at the reference laboratory for PNLAC, ANLIS in Buenos Aires, Argentina (see photo). In the paper, we show how locally-generated sequencing data from this facility can be integrated into the regional surveillance framework to determine whether outbreaks were due to locally-circulating lineages or resulted from the importation of new sublineages.

In addition to laboratory capacity building, the collaboration involved training an ANLIS researcher (Josefina Campos – see photo – who now runs the genomics facility there) in bioinformatics, and conducting training courses (in conjunction with Wellcome Trust Advanced Courses) for medical, veterinary and public health professionals in the region, including courses in Argentina, Uruguay and Costa Rica (see picture).

There are 29 authors on our paper and every one of them worked hard on, and cared deeply about, the outcome of the study as well as the training programs and capacity building surrounding it. Every paper has a story behind it, and this one, like so many others, is so much more than it appears.

Photo: Top ANLIS in Buenos Aires, Argentina. Bottom (from right to left) ANLIS collaborator Josefina Campos and co-corresponding author Nicholas Thomson (WTSI) outside the Malbran (ANLIS) Institute; Genomics for Epidemiology and Surveillance of Bacterial Pathogens course instructors and participants held in February 2015 in San Jose, Costa Rica; co-corresponding author Kate Baker with bust of Carlos Gregorio Malbran, the ANLIS institute’s namesake.