TEM of VEGF and Scrambled Control SmartFlares

The Uptake Control SmartFlare was opened first to help us to establish a loading protocol. As such, the physical characterisation of that SmartFlare was performed in an earlier post. Thanks once again go to Joan Comenge for taking the VEGF and Scrambled control SmartFlare to the Transmission Electron Microscope (TEM).

2015-05-05-TEM2

Protocol

10 μl droplets of the sample were drop casted onto a formvar/carbon filmed 200-mesh copper grid (ordered from TAAB) and left to dry in air. Gold nanoparticles were visualized using a Tecnai G3 spirit TEM at 120 KeV.

Results

All of the data can be viewed on our Open Data Repository date stamped [2015-05-05].

TEM of the Scrambled Control SmartFlare
TEM of the Scrambled Control SmartFlare

Using the same calibration and analysis protocol as previously, the nanoparticles can be measured and a distribution of sizes can be calculated. Once again we can calculate the distribution of the Major and Minor elliptical axis as a proxy for mean particle diameter:

Estimate of the mean Major Elliptical axis for VEGF (left) and Scrambled (right) SmartFlares.
Estimate of the mean Major elliptical axis for VEGF (left) and Scrambled (right) SmartFlares.

 

Estimate of the mean Minor elliptical axis for VEGF (left) and Scrambled (right) SmartFlares.
Estimate of the mean Minor elliptical axis for VEGF (left) and Scrambled (right) SmartFlares.

The Circularity is calculated as (4π*area)/(perimeter2) where a perfect circle will have a value of one and values less than this describe a more elongated shape.

Estimate of the mean Major elliptical axis for VEGF (left) and Scrambled (right) SmartFlares
Estimate of the mean Circularity for VEGF (left) and Scrambled (right) SmartFlares

Summary

The gold particles appear to be slightly smaller than the Uptake Controls (especially the VEGF SmartFlares). The circularity is basically the same.

Not being hugely familiar with EM, I’m not sure what to make of the grey dappled areas around the VEGF and to a lesser extent the Scrambled SmartFlares (click to go to the full image). Answers on a postcard (or in the comments!)

 2015-05-05-TEM3

Authors: Dave Mason & Joan Comenge

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6 thoughts on “TEM of VEGF and Scrambled Control SmartFlares

  1. Is the sample in culture media? The grey areas look like proteins to me. I have seen this with some of my samples in the past. Anita

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    • Hi Anita, the SmartFlares are resuspended in nuclease-free de-ionised water then directly dropped onto a grid to dry, so there’s no culture media. It does make me wonder if the oligos are dissociating and aggregating though.

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  2. Maybe small salt or sugar crystals?
    You might have resuspended it in de-ionised water, but it must have been lyofilized from a high-salt buffer with possible sugars to get a nice pellet.

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    • Hi Ron. That sounds like a possibility. Odd that we didn’t see the same pattern with the Uptake Controls though. I wonder how much difference in stability there is between nucleotide-conjugated fluorophores (as in the Uptake Controls) and hybridised nucleotides (like the Scrambled and VEGF SmartFlares).

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  3. One of the difficulties with working with commercial products, is that it is hard to know what they contain. Are you sure that Uptake Controls are lyophilized in exactly the same buffer as the Smartflare? It would make sense to make controls in the same buffer, but on the other hand, the controls which are not double stranded do not need the high salt concentration, and therefore they might be lyophilized from a low salt buffer.

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    • Unfortunately, we have no idea in what the original SmartFlares were lyophilised. I’ll drop an email to the company and see if they have any comment.

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