Fabricating chambers for fluid-phase FCS measurements

I previously tweeted about fabricating chambers for Fluorescent Correlation Spectroscopy (FCS). Given the response, I thought I would give a little more detail in a post.

From Wikimedia Commons

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Quantum Dot Labelling

Reproducibility is key to scientific research. It is, in fact, a hallmark of the Scientific Method, controlling variables until the same result can be achieved time again. There are few things more frustrating than following a simple experimental protocol and not achieving the predicted result. This inevitably leads to wasted time, wasted reagents, and very frustrated researchers.

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Targeting Nanoparticles to the Nucleus

Nuclear Localisation Signal (NLS) is a peptide sequence that tags certain proteins for nuclear transport. By conjugating NLS to gold nanoparticles (GNPs), this should (in theory) result in the GNPs being transported to the nucleus. This can then be detected using photothermal microscopy. However, actually putting this into practice is slightly harder. Due to the abundance of positively charged residues in the NLS sequence they can cause the nanoparticles to aggregate.

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Striking Gold(nanoparticles)!

Following the preparation of 10nm GNPs conjugated to CALNN, the next step was to use these for microinjection and then photothermal imaging. For this I used 35mm gridded dishes, this meant that the coordinates of successfully injected cells could be recorded to make the cells easier to find again. The length of time the cells were removed from the incubator had to be kept to a minimum as after extended time periods they would begin to die. After the cells had been injected they were then fixed using paraformaldehyde and stored in the fridge until needed for imaging.

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Update: we have submitted a Letter to PNAS as a response to the paper discussed below. It is currently available as a preprint.

The Mirkin group recently published the latest paper to use Spherical Nucleic Acid (SNA) technology that we’ve come to know and love as Smart/Nano Flares. In an interesting twist, instead of having target oligos bind to the nanoparticle (displacing fluorescent reporter sequences), the reporter sequences are now complimentary to the target oligos, labelling them directly. In theory this allows for dynamic labelled, tracking and quantitation of mRNA in cells.

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Imaging with Gold

Gold nanoparticles (GNPs, also known as “colloidal gold“) have become the subject of large amounts of research in recent years which can mainly be attributed to their unique properties. Their potential applications range from targeted drug delivery to cell imaging. It is their use in cell imaging, specifically photothermal microscopy, that I’m interested in for my summer project.

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Probing for Caveosomes

As mentioned before, one of the things that has been bugging me about the SmartFlares is that they’re not where anyone expects them. EMD expect them in the cytosol (we don’t see them there), I expected them in endosomes and they weren’t really there either. So what could this mystery compartment be?

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