Throughout all of the fluorescent microscopy experiments, we’ve been seeing SmartFlare fluorescence in punctate structures. Using these techniques, it’s very difficult to say with certainty that the SmartFlares are within vesicles or (for example) aggregated/agglomerated in the cytosol. As this is pertinent to the current investigation, we took cells loaded with SmartFlares to the electron microscope which has the resolution to discern membrane structures as well as the gold particles.
The protocol was essentially identical to all previous experiments, with several alterations:
- HeLa cells were grown in 35mm polystyrene dishes (cf. glass bottom dishes previously) and exposed to VEGF or Uptake Control SmartFlares as before.
- The samples were prepared for EM using the following protocol:
- After 18h incubation, cells were fixed with a solution containing 1% paraformaldehyde and 3% gluteraldehyde in 0.1 M cacodylate buffer (pH 7.4).
- First, reduced osmium staining with 2% OsO4 + 1.5% K4[Fe(CN)6]. This was followed by a 2nd osmium staining (2% OsO4) and a uranyl acetate (1%) staining.
- Samples were then dehydrated in graded ethanol (30%, 50%, 70%, 90% and 2x 100%).
- Finally, samples were infiltrated with medium TAAB resin 812 and embedded with the same resin. The resin was cured for 48h at 60°C.
- Ultrathin sections of 350μm x 350μm x 74 nm were cut and placed in 200 mesh Formvar/Carbon filmed grids. They were post-stained with uranyl acetate and lead citrate before TEM imaging.
Cells were imaged on an Tecnai G3 spirit. The data are date stamped [2015-07-14] and available on our Open Data repository. The Uptake controls will be added shortly, for the time being all of these results pertain to the VEGF SmartFlares.
Cells were imaged first at low magnification (~2900x)
And also at higher (~6800x) magnification (in the same area).
During imaging, the following observations were made:
- All SmartFlare signal observed, appeared to be within membrane-contained vesicles.
- Some cell slices did not have any SmartFlares
- There was some variability in the number of vesicles per slice. Some had more, some had fewer.
- The SmartFlare-containing vesicles were approximately 300-500nm in diameter. Many were non-uniformly shaped.
These findings support our fluorescence microscopy data, specifically the observation of cellular heterogeneity in SmartFlare uptake. Even though the data are not exhaustive, they are highly suggestive when viewed in combination with our other work.
Authors: Joan Comenge & Dave Mason