Single molecule FISH is currently the best method to get accurate measurements of mRNA levels at single molecule, single cell level in cell culture or tissue slices with a spatial resolution of ~200 nanometer (or less). One of the drawbacks of this method is the deterioration of the fluorescent signal (bleaching) of the organic dyes that are used to label the probes. Andrew Smith’s lab from University of Illinois now show how FISH can work with quantum dots instead of organic dyes. This provides better fluorophore stability and also the possibility to have more colors with less overlap of the emission spectra.
Quantum dots (QD) are nanocrystals with fluorescent properties that have been used for both fixed and live labeling of biological samples (e.g. by conjugation to specific antibodies). However, QD were not used successfully in FISH protocol. Smith suspected that the poor labeling of past QD-FISH was due to the fact that commercially available QD have relatively large sizes of 15-35 nm (compared to organic dyes which are <10 nm). This large size prevent the QD-oligo probe to get to the mRNA in the packed environment of the proteins and other molecules in cell. So they made smaller QD, and this time it works.
In this short paper they show that the FISH works as well as organic dye-FISH in detecting cytoplasmic mRNAs, but that the FISH signal is stronger and much more stable than the organic dye. Quite amazing to see the organic dye signal fade to 50% in 3 min exposure and to a close to 0% after 12 min, whereas the QD signal is stable up to 12 min. There is a clear difference between FISH with QD of 17.4 nm (low counts) to 13.3 nm (comparable to organic dye-FISH). They also show multiplexing of FISH for three colors with QD608, QD693 and QD800. It’s too bad they didn’t try to squeeze in a few more colors, because in their introduction they say that regular FISH is limited to 3 colors. So, please, show us more with QD.
One thing that strikes me a little odd is that all their FISH images lack transcription sites in the nucleus. I don’t know if this is just by chance or on purpose, but I wonder how well QD-FISH can be used to quantify transcription compared to regular FISH. Can the QD-oligo make its way through the more crowded nucleus? This is an important point if we want this new technology to be useful (and we do want it!).
Another thing I wish they would check is how well this QD-FISH survives confocal microscopy. Because it will be awesome if it does.
Last, I wonder if this can be used for electron microscopy, to get really high-resolution of the mRNA localization & structure.
Yang Liu, Phuong Le, Sung Jun Lim, Liang Ma, Suresh Sarkar, Zhiyuan Han, Stephen J. Murphy, Farhad Kosari, George Vasmatzis, John C. Cheville & Andrew M. Smith (2018) Enhanced mRNA FISH with compact quantum dots. Nature Communications 9: 4461